Genetic construct

ABSTRACT

The invention provides genetic constructs and recombinant vectors comprising such constructs. The constructs and vectors can be used in gene therapy methods for treating a range of disorders, including glaucoma and deafness, or for promoting nerve regeneration and/or survival.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/762,089, filed on Mar. 21, 2018, which is a § 371 national phase of International Application No. PCT/GB2016/053319, filed on Oct. 25, 2016, which claims the benefit of United Kingdom Application No. 1518911.1, filed on Oct. 26, 2015, which applications are incorporated by reference herein.

The present invention relates to genetic constructs and recombinant vectors comprising such constructs, and to the uses of the constructs and vectors in gene therapy methods for treating a range of disorders, including glaucoma and deafness, or for promoting nerve regeneration and/or survival.

Glaucoma is a term used to define a group of ocular disorders characterised by progressive optic nerve degeneration, death of retinal ganglion cells (RGC) and axon loss, which results in an excavated appearance of the optic nerve head and loss of vision. Glaucoma is a leading cause of blindness worldwide [1] and the incidence of glaucoma increases dramatically with age. Around half a million people in the U.K. and more than 2.2 million people in North America aged 40 and older have glaucoma. Moreover, every hour, someone goes blind from this sight-threatening disease in the U.S. [2]. As the size of the elderly population continues to grow rapidly, glaucoma has become an imminent social as well as medical problem. Elevated intraocular pressure (IOP) is the most important risk factor for glaucoma [3], besides age and all currently licensed treatments work by lowering IOP [4-5].

Glaucoma can be diagnosed prior to loss of vision by visual field testing and by ophthalmoscopic examination of the optic nerve to detect “cupping”. Current management of glaucoma is based on lowering the IOP to normal levels, which are between to and 21 mm Hg, in order to prevent further optic nerve damage using topically applied drugs [6]. The mean IOP in normal adults is 15 to 16 mm Hg. Currently there are five major classes of medications that are used to lower the IOP: β-adrenergic antagonists, adrenergic agonists, parasympathomimetics, prostaglandin-like analogues and carbonic anhydrase inhibitors [7]. Whilst relatively effective in reducing IOP when correctly used, these drugs can cause severe side effects in some patients and thereby adversely affect the quality of the patient's life. In addition, adherence to IOP-lowering eye drop treatment is often poor, particularly in elderly patients who are required to take multiple medications. It has been estimated that less than 50% of patients prescribed IOP lowering treatment actually use it regularly as directed, with obvious implications for control of the underlying condition. If additional lowering of IOP is indicated, or if medication fails to sufficiently lower the IOP, laser trabeculoplasty may be used, but this treatment fails to achieve adequate IOP lowering in many patients. If IOP is still not adequately controlled, incisional glaucoma surgery may be indicated. However, IOP lowering treatment fails to prevent deterioration in many patients and glaucoma remains the leading cause of irreversible blindness worldwide. Neuroprotection of the glaucomatous RGCs and their axon projections, which form the optic nerve, would therefore be a valuable therapeutic paradigm for use as an adjunct to conventional IOP lowering treatments and particularly important in patients deteriorating despite conventional therapy [8].

Glaucomatous optic neuropathy appears to result from specific pathophysiological changes and subsequent death of RGCs and their axons. The process of RGC death is thought to be biphasic, i.e. a primary injury responsible for initiation of damage followed by a slower, secondary degeneration attributable to the hostile environment surrounding the degenerating cells [9].

RGC death mechanisms in experimental animal models of glaucoma and human glaucoma have been shown to involve apoptosis [10]. Although the molecular mechanism triggering apoptosis has not been identified, deprivation of neurotrophic factors, ischemia, chronic elevation of glutamate and disorganized nitric oxide metabolism are suspected to be possible mechanisms [11].

Brain-derived neurotrophic factor (BDNF) along with nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are members of the neurotrophin family of trophic factors [12-13]. The neurotrophins play essential roles in the development, survival and function of a wide range of neurons in both the peripheral and central nervous systems, including RGCs. Neurotrophins interact with two cell surface receptors, low affinity p75^(NTR) receptors and the high affinity tyrosine receptor kinase (Trk) family [12-13]. Nerve growth factor (NGF) preferentially binds TrkA, Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-4/5 (NT4/5) bind to tropmyosin receptor kinase-B (TrkB), and Neurotrophin-3 (NT-3) binds TrkC (and TrkA to a lesser extent) [12-13].

Among neurotrophins, BDNF is the most potent survival factor for injured RGCs [14-21]. BDNF is a protein molecule produced in the brain and transported to the retina by way of retrograde axonal transport through the optic nerve, where it supports RGCs and maintains their survival [15-21]. In certain conditions, such as during excitotoxic insults with glutamate receptor agonists, such as N-methyl-D-aspartate, BDNF can also be produced in RGCs although at relatively low levels [22-23]. BDNF is normally produced as a prepro-polypeptide (i.e. preproBDNF) containing a short signal peptide sequence, which facilitates trafficking of the entire polypeptide to vesicles for release into the extracellular space. Cleavage and removal of the signal peptide converts preproBDNF into proBDNF. An N-terminal proBDNF sequence is then cleaved either intracellulary or extracellularly to create mature BDNF (mBDNF) [24]. Both pro-BDNF and mBDNF possess biological activity with pro-BDNF preferentially activating p75^(NTR) receptors and the shorter mBDNF activating TrkB receptors [25-27]. Activation of P75^(NTR) and TrkB receptors in the retina show opposing effects on RGC survival, the former being responsible for apoptosis through direct RGC-cell-body-p75^(NTR)-activation [25-28] or indirectly via p75^(NTR) activation on Müller cells, thereby stimulating release of Tumour Necrosis Factor-alpha (TNF-α) which further promotes RGC loss [29].

Animal models of glaucoma have demonstrated that following nerve crush, or raised IOP, there is a shift away from neurotrophic mBDNF/TrkB signalling towards pro-BDNF/p75^(NTR) pathways. Reduced levels of mBDNF and TrkB receptors in the retina have been demonstrated [27, 30-31] together with opposing elevations in the relative levels of pro-BDNF [28] and p75^(NTR) receptors [32]. Supplementation of mBDNF through ocular injections of recombinant protein to rats with experimentally elevated IOP increases the survival of RGCs compared with untreated eyes, thereby confirming a key neuroprotective role for this neurotrophin [19-21].

To maintain levels of mBDNF in eyes with glaucoma, regular injections of mBDNF would be required as mBDNF is rapidly degraded within the eye. To overcome the need for regular intraocular injections of mBDNF, attempts to provide constant elevated BDNF have resorted to using recombinant adenovirus or adeno-associated viral (rAAV) vector delivery of the transgene coding for BDNF to the retina to delay or prevent RGC death in animal models of glaucoma [18, 33-34]. rAAV vectors consist of a single-stranded DNA genome. They have has been successfully used as a viral vector for gene therapy in multiple clinical trials whilst displaying limited toxicity. Whilst intravitreal injections of recombinant mBDNF alone, or increasing local BDNF production via gene therapy, have been shown to be effective in preventing loss in RGCs over a short period following IOP elevation or other optic nerve damage, the beneficial effect of BDNF have been shown to be transient [18]. However, gene therapy which incorporates the endogenous BDNF gene sequence is also capable of producing and releasing pro-BDNF as well as the intended mBDNF.

Gene therapy aimed at attenuating or preventing loss in TrkB signalling through increased expression of the receptor in RGCs or through constant stimulation of remaining extracellular TrkB receptors using an antibody with agonist properties has also demonstrated success in preventing RGC loss [35-36]. However, reductions in trophic signalling through the mBDNF/TrkB pathway is further complicated by internalisation of mBDNF-activated TrkB receptors and replacement of these receptors at the cell surface with TrkB isoforms incapable of intracellular signalling [37-38]. Furthermore, the biochemical system responsible for deactivation of TrkB receptors following autophosphorylation of TrkB receptor dimers in the presence of mBDNF is upregulated in retinas subjected to raised IOP [39].

Furthermore, in addition to glaucoma, the BDNF/TrkB axis has also been implicated in neuroprotection of components of the inner ear, specifically of the cochlear structure where insults can result in loss of hair cells resulting in deafness [40-42], and of nerve regeneration [43-44].

There is therefore a need for an improved gene therapy for the treatment of glaucoma and deafness, and for promoting nerve regeneration or survival.

The inventors have constructed a novel genetic construct, which encodes the tyrosine kinase receptor B (TrkB), and an agonist of the TrkB receptor under the control of a single promoter. The promoter of the construct may be used to ensure that the agonist and the receptor are only expressed in retinal ganglion cells (RGCs), cochlear or nerve cells, and promote the survival of these cells.

Thus, according to a first aspect of the invention, there is provided a genetic construct comprising a promoter operably linked to a first coding sequence, which encodes the tyrosine kinase receptor B (TrkB), and a second coding sequence, which encodes an agonist of the TrkB receptor.

The inventors have demonstrated in the Examples that it is possible to combine the genes which code for both the TrkB receptor and its agonist in a single genetic construct. This was especially challenging given their large sizes, and it could not have been predicted that it would have been possible to co-express them in physiologically useful concentrations. Advantageously, with the construct of the invention, there is no need to inject a recombinant protein, as described in the prior art. Furthermore, in the prior art, it is still necessary to perform regular injections of protein, whereas the construct of the invention only requires a single gene therapy injection.

Preferably, in use, the TrkB receptor is activated by the agonist to thereby promote survival of retinal ganglion cells (RGCs), nerve cells or cochlear cells. Advantageously, the construct of the invention may therefore be used to target RGCs, nerve cells or cochlear cells in order to maintain or enhance TrkB-signalling in these cells. Thus, the construct may be used to maximise protection against pathophysiological stressors of glaucoma and deafness, and to promote nerve regeneration and/or survival. Furthermore, the construct may be used to provide long-term treatment of glaucoma or deafness due to the expression of the TrkB receptor and an agonist of the receptor under the control of one or more promoter. Consequently, the construct has overcome the need to use multiple alternative treatments, which, even in combination, provide a transient therapeutic effect. Moreover, the construct of the invention is advantageous because it may be used to significantly enhance RGC or cochlear cell sensitivity to TrkB receptor agonists due to a localised increase in both the TrkB receptor and the agonist of the receptor.

Preferably, the genetic construct of the invention comprises an expression cassette, one embodiment of which is shown in FIG. 1. As can be seen in FIG. 1, the construct comprises the promoter, the first nucleotide sequence encoding the TrkB receptor, and the second nucleotide sequence encoding mature brain derived neurotrophic (mBDNF), which acts as a preferred agonist of the TrkB receptor. It will be appreciated, however, that other agonist may be used, as discussed herein. Also as shown in FIG. 1, the expression cassette also includes a 2A spacer sequence, a sequence encoding Hepatitis Virus Post-transciptional Regulatory Element (WHPE), a sequence encoding a polyA tail, and left and right hand Inverted Terminal Repeat sequences (ITRs).

Hence, preferably the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, which spacer sequence encodes a peptide spacer that is configured to be digested or cut to thereby produce the TrkB receptor and the agonist as separate molecules. In the embodiment illustrated in FIG. 1, the coding sequence for the TrkB receptor is disposed 5′ of the coding sequence for the receptor agonist (BDNF) with the spacer sequence therebetween. However, in another embodiment, the coding sequence for the receptor agonist may be disposed 5′ of the coding sequence for the receptor with the spacer sequence therebetween.

Preferably, the genetic construct comprises a nucleotide sequence encoding Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WHPE), which enhances the expression of the two transgenes, i.e. the TrkB receptor and its agonist, which is preferably BDNF. Preferably, the WHPE coding sequence is disposed 3′ of the transgene coding sequence.

One embodiment of the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WHPE) is 592 bp long, including gamma-alpha-beta elements, and is referred to herein as SEQ ID No: 57, as follows:

[SEQ ID NO. 57] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGT ATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAA TCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACG TGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCA TTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCT ATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGG GGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGA CGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGG ACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTC CCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCC CTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG

Preferably, the WHPE comprises a nucleic acid sequence substantially as set out in SEQ ID No: 57, or a fragment or variant thereof.

However, in a preferred embodiment, a truncated WHPE is used, which is 247 bp long due to deletion of the beta element, and which is referred to herein as SEQ ID No: 58, as follows:

[SEQ ID NO. 58] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGT ATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAA TCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCG CTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGT

Advantageously, the truncated WHPE sequence used in the construct saved about 300 bp in total without negatively impacting on transgene expression. Preferably, the WHPE comprises a nucleic acid sequence substantially as set out in SEQ ID No: 58, or a fragment or variant thereof.

Preferably, the genetic construct comprises a nucleotide sequence encoding a polyA tail. Preferably, the polyA tail coding sequence is disposed 3′ of the transgene coding sequence, and preferably 3′ of the WHPE coding sequence.

Preferably, the polyA tail comprises the simian virus 40 poly-A 224 bp sequence. One embodiment of the polyA tail is referred to herein as SEQ ID No: 59, as follows:

[SEQ ID NO. 59] AGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAA TGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTA TTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCAT TCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCA AGTAAAACCTCTACAAATGTGGTA

Preferably, the polyA tail comprises a nucleic acid sequence substantially as set out in SEQ ID No: 59, or a fragment or variant thereof.

Preferably, the genetic construct comprises left and/or right Inverted Terminal Repeat sequences (ITRs). Preferably, each ITR is disposed at the 5′ and/or 3′ end of the construct.

The promoter in the genetic construct of the first aspect may be any nucleotide sequence that is capable of inducing RNA polymerase to bind to and transcribe the first and second coding sequences. In one preferred embodiment, the promoter is the human synapsin I (SYN I) promoter. One embodiment of the 469 nucleotide sequence encoding the human synapsin I (SYN I) promoter is referred to herein as SEQ ID NO.1, as follows:

[SEQ ID NO. 1] CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGA GGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAG CACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAG GGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGC GGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGC ACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCC CGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACC ACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGC GCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGT GTCGTGCCTGAGAGCGCAG

Preferably, therefore, the promoter may comprise a nucleotide acid sequence substantially as set out in SEQ ID No: 1, or a fragment or variant thereof.

In another preferred embodiment, the promoter is the CAG promoter. The CAG promoter preferably comprises the cytomegalovirus early enhancer element, the first exon and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene, thereby facilitating tissue specific expression in RGCs and cochlear cells only. One embodiment of the 1733 nucleotide sequence encoding the CAG promoter is referred to herein as SEQ ID NO.2, as follows:

[SEQ ID NO. 2] CTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCA TTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAA TGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAA TGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG TACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCAC GTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGT ATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGG GGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGC GAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGC GCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTC CGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCC CACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGC TTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGA GGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGT GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGC GGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGT GCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCT GCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCA GGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCC CCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGC GTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTG CCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCG CGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCC ATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCC CAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAG CGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGG AGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCT CGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGC GGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACC ATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTT ATTGTGCTGTCTCATCATTTTGGCAAAGAATTG

In another preferred embodiment, the promoter is a truncated form of the CAG promoter, such as a 664 nucleotide form of the promoter referred to herein as SEQ ID NO.3, as follows:

[SEQ ID No: 3] CTAGATCTGAATTCGGTACCCTAGTTATTAATAGTAATCAATTACGGGGT CATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTA AATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAT AATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTG TATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCC CGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGC AGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCC ACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTT GTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGG GGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGG GCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAA GTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAA GCGCGCGGCGGGCG

In yet a further preferred embodiment, the promoter is a truncated form of the CAG promoter, such as a 584 nucleotide form of the promoter referred to herein as SEQ ID NO. 48, as follows:

[SEQ ID No: 48] GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAAT AGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCC ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA CCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCC CCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGC GGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAAT CAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCG GCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

Therefore, preferably the promoter comprises a nucleotide acid sequence substantially as set out in SEQ ID No: 2, 3 or 48, or a fragment or variant thereof.

Many bicistronic gene constructs presented in the scientific literature have either (i) incorporated dual promoters to separately drive expression of two genes, or (ii) use the internal ribosome entry site (IRES) of the encepahlomyocarditis virus (EMCV) to link two genes transcribed from a single promoter within recombinant viral vectors [45-46]. However, the efficiency of IRES-dependent translation may vary in different cells and tissues and IRES-dependent second gene expression can be significantly lower than cap-dependent first gene expression in bicistronic vectors [47]. Moreover, the size limitation of rAAV vectors (generally <5 kb) will prevent the incorporation of large gene constructs, such as the TrkB receptor together with BDNF using dual promoters or IRES linkers.

Accordingly, in a preferred embodiment, the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, which spacer sequence encodes a peptide spacer that is configured to be digested to thereby produce the TrkB receptor and agonist as separate molecules. Preferably, the spacer sequence comprises and encodes a viral peptide spacer sequence, more preferably a viral 2A peptide spacer sequence [47]. Preferably, the 2A peptide sequence connects the first coding sequence to the second coding sequence. This enables the construct to overcome the size restrictions that occur with expression in various vectors and enables expression of all of the peptides encoded by the construct of the first aspect to occur under control of a single promoter, as a single protein.

Thus, following the translation of the single protein containing the sequences of TrkB, the 2A peptide, and the agonist (preferably BDNF), cleavage occurs in the viral 2A peptide sequence at the terminal glycine-proline link, thereby liberating two proteins, i.e. TrkB and agonist (i.e. mBDNF). The genetic construct is designed such that the remaining short N-terminal amino acid sequence of the viral 2A peptide remain attached to the intracellular portion of the TrkB receptor, thereby removing immunogenicity risks and not interfering with the intracellular signalling capability of the mature receptor. The residual proline amino acid from the C-terminal viral 2A sequence remains attached to the N-terminal BDNF signal peptide and is ultimately removed from the mBDNF protein following cleavage of the signal sequence from the mature protein.

The inventors have generated two embodiments of the spacer sequence. One important section of the peptide spacer sequence, which is common to both embodiments described herein, is the C-terminus. Accordingly, preferably the peptide spacer sequence comprises an amino acid sequence referred to herein as SEQ ID NO. 4, or a fragment or variant thereof, as follows:

[SEQ ID No: 4] QAGDVEENPGP

Preferably, the digestion or cut site of the peptide spacer sequence is disposed between the terminal glycine and end proline in SEQ ID No:4.

In a first preferred embodiment, the spacer sequence comprises a nucleotide sequence referred to herein as SEQ ID NO.5, or a fragment or variant thereof, as follows:

[SEQ ID No: 5] GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGGCTGGAGACGTGGAGGA GAACCCTGGACCT

In this first embodiment, the peptide spacer sequence comprises an amino acid sequence referred to herein as SEQ ID NO. 6, or a fragment or variant thereof, as follows:

[SEQ ID No: 6] GSGATNFSLLQAGDVEENPGP

In a second preferred embodiment, the spacer sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 7, or a fragment or variant thereof, as follows:

[SEQ ID No: 7] AGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGA GAACCCTGGACCT

In this second embodiment, the peptide spacer sequence comprises an amino acid sequence referred to herein as SEQ ID NO. 8, or a fragment or variant thereof, as follows:

[SEQ ID No: 8] SGATNFSLLKQAGDVEENPGP

The inventors have carefully considered the sequences of the TrkB receptor, and have produced several preferred embodiments of the receptor that is encoded by the first coding sequence in the genetic construct of the first aspect.

In one preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding the human canonical isoform of TrkB. Preferably, the canonical isoform of TrkB comprises an amino acid sequence (822 residues) referred to herein as SEQ ID NO. 9, or a fragment or variant thereof, as set out below:

[SEQ ID No: 9] MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSP GIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVD SGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPF TCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSAN LAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSL RITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHH WCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDN PTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDY GTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVM LFLLKLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDA VIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFG KVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHI VKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQ SQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRD VYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYG KQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNI KGIHTLLQNLAKASPVYLDILG

Preferably, in this embodiment, the first coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 10, or a fragment or variant thereof, as set out below:

[SEQ ID No: 10] ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGG CTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCA CGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCT GGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAA CATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACG AAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGAT TCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCT GCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAAC ATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTT ACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATC CAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATA TTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAAT CTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATC CTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTA ACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTA AGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGT GGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGC ATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCAC TGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTG GTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAA TACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAAT CCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTA TGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAA TTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTAT GGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAAT CCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCT ATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATG CTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGGCCC AGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACA TCTCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCT GTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGTACTT TGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCA AGCGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGA AAAGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGAT CTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGG ACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATC GTCAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTT TGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCC CTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAG TCGCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCT GGCGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGG TCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGAC GTGTACAGCACTGACTACTACAGGGTCGGTGGCCACACAATGCTGCCCAT TCGCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAA GCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGC AAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCAC TCAGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATG AGCTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATC AAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCTA CCTGGACATTCTAGGC

In another preferred embodiment, the first coding sequence comprises a nucleotide sequence which encodes isoform 4 of TrkB. Preferably, isoform 4 of TrkB comprises an amino acid sequence referred to herein as SEQ ID NO. 11, or a fragment or variant thereof, as set out below:

[SEQ ID No: 11] MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSP GIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVD SGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPF TCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSAN LAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTOGSL RITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHH WCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDN PTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDY GTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVM LFLLKLARHSKFGMKDFSWFGFGKVKSRQGVGPASVISNDDDSASPLHHI SNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIK RHNIVLKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKD FHREAELLTNLQHEHIVKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGP DAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLV GENLLVKIGDFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTES DVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYE LMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG

Preferably, this embodiment of the first coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 12, or a fragment or variant thereof, as set out below:

[SEQ ID No: 12] ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGG CTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCA CGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCT GGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAA CATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACG AAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGAT TCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCT GCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAAC ATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTT ACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATC CAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATA TTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAAT CTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATC CTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTA ACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTA AGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGT GGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGC ATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCAC TGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTG GTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAA TACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAAT CCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTA TGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAA TTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTAT GGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAAT CCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCT ATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATG CTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGATTT CTCATGGTTTGGATTTGGGAAAGTAAAATCAAGACAAGGTGTTGGCCCAG CCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACATC TCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCTGT CATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGTACTTTG GCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCAAG CGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGAAA AGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGATCT TGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGGAC TTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATCGT CAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTTTG AGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCCCT GATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAGTC GCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCTGG CGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGGTC GGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGACGT GTACAGCACTGACTACTACAGGGTCGGTGGCCACACAATGCTGCCCATTC GCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAAGC GACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGCAA ACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCACTC AGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATGAG CTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATCAA GGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCTACC TGGACATTCTAGGC

The inventors have spent considerable inventive endeavour in studying the sequence of the TrkB receptor and have realised that TrkB comprises five tyrosine residues (at position 516, 701, 705, 706 and 816 of SEQ ID No: 9), which are normally phosphorylated following dimerization and autophosphorylation in the presence of a BDNF dimer. A problem with phosphorylation of these five tyrosine residues is that the receptor can be readily deactivated by a phosphatase, such as the Shp-2 phosphatase. Accordingly, in order to prevent phosphorylation and resultant deactivation of the receptor in vivo, preferably one or more of these key tyrosines is mutated (more preferably, to glutamic acid) in order to mimic the resultant phosphotyrosine and produce a receptor which remains active in the presence of BDNF, and which cannot be deactivated by a phosphatise, such as the Shp-2 phosphatase. Such mutant forms of TrkB are aimed at producing TrkB receptor activity which remains active for longer periods, or until the receptor is internalised.

The DNA and amino acid sequences provided below illustrate the positions of these five tyrosine (Y) residues which have been mutated into five glutamic acid (E) residues. It will be appreciated that 1, 2, 3, 4 or 5 of these residues may be mutated to glutamic acid in embodiments of the invention. Various combinations of these mutations is also envisaged, e.g. positions 516 and 701 only, or positions 705, 706 and 816 only, and so on.

Accordingly, in another preferred embodiment, the first coding sequence comprises a nucleotide sequence encoding a mutant form of TrkB receptor, wherein one or more tyrosine residue at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 is modified or mutated. Preferably, at least two, three or four tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified. Most preferably, all five tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified.

Preferably, the or each tyrosine residue is modified to a different amino acid residue, more preferably a glutamic acid. Thus, preferably the mutant form of the TrkB receptor comprises Y516E, Y701E, Y705E, Y706E and/or Y816E.

Preferably, the modified form of the TrkB receptor comprises an amino acid sequence referred to herein as SEQ ID NO. 13, or a fragment or variant thereof, as set out below:

[SEQ ID No: 13] MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSP GIVAFPRLEPNSVDPENITEIFIANQKRLEIINEDDVEAYVGLRNLTIVD SGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPF TCSCDIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSAN LAAPNLTVEEGKSITLSCSVAGDPVPNMYWDVGNLVSKHMNETSHTQGSL RITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHH WCIPFTVKGNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDN PTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGIDDGANPNYPDVIYEDY GTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVM LFLLKLARHSKFGMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDA VIIGMTKIPVIENPQEFGITNSQLKPDTFVQHIKRHNIVLKRELGEGAFG KVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHI VKFYGVCVEGDPLIMVFEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQ SQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGDFGMSRD VESTDEERVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYG KQPWYQLSNNEVIECITQGRVLQRPRTCPQEVYELMLGCWQREPHMRKNI KGIHTLLQNLAKASPVELDILG

Preferably, in this embodiment, the first coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 14, or a fragment or variant thereof, as set out below:

[SEQ ID No: 14] ATGTCGTCCTGGATAAGGTGGCATGGACCCGCCATGGCGCGGCTCTGGGG CTTCTGCTGGCTGGTTGTGGGCTTCTGGAGGGCCGCTTTCGCCTGTCCCA CGTCCTGCAAATGCAGTGCCTCTCGGATCTGGTGCAGCGACCCTTCTCCT GGCATCGTGGCATTTCCGAGATTGGAGCCTAACAGTGTAGATCCTGAGAA CATCACCGAAATTTTCATCGCAAACCAGAAAAGGTTAGAAATCATCAACG AAGATGATGTTGAAGCTTATGTGGGACTGAGAAATCTGACAATTGTGGAT TCTGGATTAAAATTTGTGGCTCATAAAGCATTTCTGAAAAACAGCAACCT GCAGCACATCAATTTTACCCGAAACAAACTGACGAGTTTGTCTAGGAAAC ATTTCCGTCACCTTGACTTGTCTGAACTGATCCTGGTGGGCAATCCATTT ACATGCTCCTGTGACATTATGTGGATCAAGACTCTCCAAGAGGCTAAATC CAGTCCAGACACTCAGGATTTGTACTGCCTGAATGAAAGCAGCAAGAATA TTCCCCTGGCAAACCTGCAGATACCCAATTGTGGTTTGCCATCTGCAAAT CTGGCCGCACCTAACCTCACTGTGGAGGAAGGAAAGTCTATCACATTATC CTGTAGTGTGGCAGGTGATCCGGTTCCTAATATGTATTGGGATGTTGGTA ACCTGGTTTCCAAACATATGAATGAAACAAGCCACACACAGGGCTCCTTA AGGATAACTAACATTTCATCCGATGACAGTGGGAAGCAGATCTCTTGTGT GGCGGAAAATCTTGTAGGAGAAGATCAAGATTCTGTCAACCTCACTGTGC ATTTTGCACCAACTATCACATTTCTCGAATCTCCAACCTCAGACCACCAC TGGTGCATTCCATTCACTGTGAAAGGCAACCCCAAACCAGCGCTTCAGTG GTTCTATAACGGGGCAATATTGAATGAGTCCAAATACATCTGTACTAAAA TACATGTTACCAATCACACGGAGTACCACGGCTGCCTCCAGCTGGATAAT CCCACTCACATGAACAATGGGGACTACACTCTAATAGCCAAGAATGAGTA TGGGAAGGATGAGAAACAGATTTCTGCTCACTTCATGGGCTGGCCTGGAA TTGACGATGGTGCAAACCCAAATTATCCTGATGTAATTTATGAAGATTAT GGAACTGCAGCGAATGACATCGGGGACACCACGAACAGAAGTAATGAAAT CCCTTCCACAGACGTCACTGATAAAACCGGTCGGGAACATCTCTCGGTCT ATGCTGTGGTGGTGATTGCGTCTGTGGTGGGATTTTGCCTTTTGGTAATG CTGTTTCTGCTTAAGTTGGCAAGACACTCCAAGTTTGGCATGAAAGGCCC AGCCTCCGTTATCAGCAATGATGATGACTCTGCCAGCCCACTCCATCACA TCTCCAATGGGAGTAACACTCCATCTTCTTCGGAAGGTGGCCCAGATGCT GTCATTATTGGAATGACCAAGATCCCTGTCATTGAAAATCCCCAGGAATT TGGCATCACCAACAGTCAGCTCAAGCCAGACACATTTGTTCAGCACATCA AGCGACATAACATTGTTCTGAAAAGGGAGCTAGGCGAAGGAGCCTTTGGA AAAGTGTTCCTAGCTGAATGCTATAACCTCTGTCCTGAGCAGGACAAGAT CTTGGTGGCAGTGAAGACCCTGAAGGATGCCAGTGACAATGCACGCAAGG ACTTCCACCGTGAGGCCGAGCTCCTGACCAACCTCCAGCATGAGCACATC GTCAAGTTCTATGGCGTCTGCGTGGAGGGCGACCCCCTCATCATGGTCTT TGAGTACATGAAGCATGGGGACCTCAACAAGTTCCTCAGGGCACACGGCC CTGATGCCGTGCTGATGGCTGAGGGCAACCCGCCCACGGAACTGACGCAG TCGCAGATGCTGCATATAGCCCAGCAGATCGCCGCGGGCATGGTCTACCT GGCGTCCCAGCACTTCGTGCACCGCGATTTGGCCACCAGGAACTGCCTGG TCGGGGAGAACTTGCTGGTGAAAATCGGGGACTTTGGGATGTCCCGGGAC GTGGAAAGCACTGACGAAGAAAGGGTCGGTGGCCACACAATGCTGCCCAT TCGCTGGATGCCTCCAGAGAGCATCATGTACAGGAAATTCACGACGGAAA GCGACGTCTGGAGCCTGGGGGTCGTGTTGTGGGAGATTTTCACCTATGGC AAACAGCCCTGGTACCAGCTGTCAAACAATGAGGTGATAGAGTGTATCAC TCAGGGCCGAGTCCTGCAGCGACCCCGCACGTGCCCCCAGGAGGTGTATG AGCTGATGCTGGGGTGCTGGCAGCGAGAGCCCCACATGAGGAAGAACATC AAGGGCATCCATACCCTCCTTCAGAACTTGGCCAAGGCATCTCCGGTCGA ACTGGACATTCTAGGC

It will be appreciated that the second coding sequence encodes an agonist of the TrkB receptor, which is preferably a member of the neurotrophin family of trophic factors. Preferred agonists of the TrkB receptor may therefore be selected from a group of agonists consisting of: Brain-derived neurotrophic factor (BDNF); nerve growth factor (NGF); neurotrophin-3 (NT-3); neurotrophin-4 (NT-4); and neurotrophin-5 (NT-5); or fragments thereof.

The nucleotide and amino acid sequences of each of these agonists will be known to the skilled person. However, by way of example, the amino acid sequence of one embodiment of Neurotrophin-4 (NT-4) is substantially as set out in SEQ ID NO. 49, as follows:

[SEQ ID No: 49] MLPLPSCSLPILLLFLLPSVPIESQPPPSTLPPFLAPEWDLLSPRVVLSR GAPAGPPLLFLLEAGAFRESAGAPANRSRRGVSETAPASRRGELAVCDAV SGWVTDRRTAVDLRGREVEVLGEVPAAGGSPLRQYFFETRCKADNAEEGG PGAGGGGCRGVDRRHWVSECKAKQSYVRALTADAQGRVGWRWIRIDTACV CTLLSRTGRA

The nucleic acid coding sequence of this embodiment of Neurotrophin-4 (NT-4) is substantially as set out in SEQ ID NO. 50, as follows:

[SEQ ID No: 50] ATGCTCCCTCTCCCCTCATGCTCCCTCCCCATCCTCCTCCTTTTCCTCCT CCCCAGTGTGCCAATTGAGTCCCAACCCCCACCCTCAACATTGCCCCCTT TTCTGGCCCCTGAGTGGGACCTTCTCTCCCCCCGAGTAGTCCTGTCTAGG GGTGCCCCTGCTGGGCCCCCTCTGCTCTTCCTGCTGGAGGCTGGGGCCTT TCGGGAGTCAGCAGGTGCCCCGGCCAACCGCAGCCGGCGTGGGGTGAGCG AAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTGCGATGCAGTC AGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGCGTGGGCGCGA GGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGTCCCCTCCGCC AGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGAGGAAGGTGGC CCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGAGGCACTGGGT ATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTGACCGCTGATG CCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACACTGCCTGCGTC TGCACACTCCTCAGCCGGACTGGCCGGGCC

The amino acid sequence of the signal peptide for the NT-4 sequence is substantially as set out in SEQ ID NO. 51, as follows:

[SEQ ID No: 51] MLPLPSCSLPILLLFLLPSVPIES

The nucleic acid sequence of this signal peptide is substantially as set out in SEQ ID NO. 52, as follows:

[SEQ ID No: 52] ATGCTCCCTCTCCCCTCATGCTCCCTCCCCATCCTCCTCCTTTTCCTCCT CCCCAGTGTGCCAATTGAGTCC

The amino acid sequence of the propeptide for this NT-4 sequence is substantially as set out in SEQ ID NO. 53, as follows:

[SEQ ID No: 53] QPPPSTLPPFLAPEWDLLSPRVVLSRGAPAGPPLLFLLEAGAFRESAGAP ANRSRR

The nucleic acid sequence of this propeptide is substantially as set out in SEQ ID NO. 54, as follows:

[SEQ ID No: 54] CAACCCCCACCCTCAACATTGCCCCCTTTTCTGGCCCCTGAGTGGGACCT TCTCTCCCCCCGAGTAGTCCTGTCTAGGGGTGCCCCTGCTGGGCCCCCTC TGCTCTTCCTGCTGGAGGCTGGGGCCTTTCGGGAGTCAGCAGGTGCCCCG GCCAACCGCAGCCGGCGT

The amino acid sequence of the mature protein sequence for this NT-4 sequence is substantially as set out in SEQ ID NO. 55, as follows:

[SEQ ID No: 55] GVSETAPASRRGELAVCDAVSGWVTDRRTAVDLRGREVEVLGEVPAAGGS PLRQYFFETRCKADNAEEGGPGAGGGGCRGVDRRHWVSECKAKQSYVRAL TADAQGRVGWRWIRIDTACVCTLLSRTGRA

The nucleic acid coding sequence of this mature NT-4 protein is substantially as set out in SEQ ID NO. 56, as follows:

[SEQ ID No: 56] GGGGTGAGCGAAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTG CGATGCAGTCAGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGC GTGGGCGCGAGGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGT CCCCTCCGCCAGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGA GGAAGGTGGCCCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGA GGCACTGGGTATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTG ACCGCTGATGCCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACAC TGCCTGCGTCTGCACACTCCTCAGCCGGACTGGCCGGGCC

Accordingly, in one preferred embodiment, the second coding sequence encodes neurotrophin-4 (NT-4), which may comprise an amino acid sequence substantially as set out in SEQ ID NO: 49 or 55, or fragment or variant thereof. Thus, the second coding sequence may comprise a nucleotide sequence substantially as set out in SEQ ID No: 50 or 56, or a fragment or variant thereof.

Most preferred agonists of the TrkB receptor, however, include prepro-brain derived neurotrophic factor (pre-pro-BDNF), pro-BDNF or mature BDNF (mBDNF). BDNF is initially synthesised as the precursor protein, preproBDNF, by ribosomes found on endoplasmic reticulum. There are at least 17 known splice variants encoded by the human preproBDNF gene (ENSG00000176697). Once preproBDNF has entered into the rough endoplasmic reticulum, preproBDNF is converted into proBDNF by cleavage of the signal peptide (i.e. the “pre” sequence). proBDNF is converted into mBDNF by cleavage of an additional N-terminal peptide sequence that is present on proBDNF. Both proBDNF and mBDNF are then secreted into the extracellular space, where they bind to and activate receptors on various cells, including RGCs and cochlear cells.

proBDNF preferentially binds to and activates the receptor, p75^(NTR), which, when activated, induces apoptosis in RGCs and cochlear cells. Thus, in one preferred embodiment, proBDNF is an agonist of the p75^(NTR) receptor. In one embodiment, the proBDNF is canonical proBDNF. Preferably, canonical proBDNF comprises an amino acid sequence referred to herein as SEQ ID NO. 15, or a fragment or variant thereof, as set out below:

[SEQ ID No: 15] APMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHVIE ELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLD AANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLE KVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRAL TMDSKKRIGWRFIRIDTSCVCTLTIKRGR

Preferably, in this embodiment, the second coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 16, or a fragment or variant thereof, as set out below:

[SEQ ID No: 16] GCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTGGCTTGGCCTACCC AGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAGGCAG GTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGATAGAA GAGCTGTTGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACAATAA GGACGCAGACTTGTACACGTCCAGGGTGATGCTCAGTAGTCAAGTGCCTT TGGAGCCTCCTCTTCTCTTTCTGCTGGAGGAATACAAAAATTACCTAGAT GCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTGACCCTGCCCGCCG AGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAG ACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAA AAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAA GTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGACAAAA GGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGCCCTT ACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACAC TTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAG

In another embodiment, the proBDNF is isoform 2 of proBDNF, which preferably comprises a Valine to methionione mutation (amino acid underlined). Preferably, isoform 2 of proBDNF comprises an amino acid sequence referred to herein as SEQ ID NO. 17, or a fragment or variant thereof, as set out below:

[SEQ ID No: 17] APMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADTFEHMIE ELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLD AANMSMRVRRHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLE KVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRAL TMDSKKRIGWRFIRIDTSCVCTLTIKRGR

In one embodiment, however, the agonist is not proBDNF, or a fragment or variant thereof, but instead the second coding sequence preferably comprises a nucleotide sequence which encodes mature BDNF. Mature BDNF (mBDNF) preferentially binds to, and activates, TrkB, which, when activated, promotes survival of RGCs and/or cochlear cells. Thus, mature BDNF is a most preferred agonist of TrkB. The construct according to the first aspect is advantageous because, unlike other known genetic constructs, the construct is capable of producing mature BDNF protein, which has not been mis-folded.

Thus, in one preferred embodiment, the second coding sequence comprises a nucleotide sequence which encodes mature BDNF. mBDNF is common to all 17 isoforms encoded by the gene. There 7 protein different sequences, five of which have extended signal sequences to the canonical form, and one has a canonical signal sequence, but a Valine to Methionine mutation (which is common to isoforms 2, 4, 7, 8, 9, 10, 11, 12, 13, 14 and 16). It is believed that the valine to methionine mutation reduces release of BDNF from the cell.

Preferably, mature BDNF comprises an amino acid sequence referred to herein as SEQ ID NO. 18, or a fragment or variant thereof, as set out below:

[SEQ ID No: 18] HSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLK QYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGW RFIRIDTSCVCTLTIKRGR

Preferably, this embodiment of the second coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 19, or a fragment or variant thereof, as set out below:

[SEQ ID No: 19] ATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTTGGTTGCATGAA GGCTGCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTGGCTTGGCCT ACCCAGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAG GCAGGTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGAT AGAAGAGCTGTTGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACA ATAAGGACGCAGACTTGTACACGTCCAGGGTGATGCTCAGTAGTCAAGTG CCTTTGGAGCCTCCTCTTCTCTTTCTGCTGGAGGAATACAAAAATTACCT AGATGCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTGACCCTGCCC GCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCG GCAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCT TGAAAAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGA CCAAGTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGAC AAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGC CCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAG ACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAG

In yet another preferred embodiment, the agonist is mBDNF with a signal peptide conjugated to its N-terminus. As discussed below, the signal peptide may be canonical signal peptide of preproBDNF, or the signal peptide of IL-2, or a de novo novel signal sequence created by the inventors.

Preferably, the second coding sequence comprises a nucleotide sequence encoding a signal peptide for the agonist of the TrkB receptor, most preferably a signal peptide for BDNF. In one preferred embodiment, the nucleotide sequence encodes the canonical signal peptide for BDNF. Preferably, this embodiment of the second coding sequence comprises a nucleotide sequence which encodes a signal peptide comprising an amino acid sequence referred to herein as SEQ ID NO. 20, or a fragment or variant thereof, as set out below:

[SEQ ID No: 20] MTILFLTMVISYFGCMKA

Preferably, this embodiment of the second coding sequence comprises a nucleotide sequence referred to herein as SEQ ID NO. 21, or a fragment or variant thereof, as set out below:

[SEQ ID No: 21] ATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAA GGCG

The inventors have created a series of extended signal peptides. In preferred embodiments, the nucleotide sequence encoding an isoform signal peptide for BDNF is selected from the group consisting of: isoform 2, 3, 6, 5 and 4. The nucleic acid and amino acid sequences for each of these extended signal peptides are set out below.

Isoform 2 [SEQ ID No: 22] MFHQVRRVMTILFLTMVISYFGCMKA [SEQ ID No: 23] ATGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTTTCCTTACTATGGT TATTTCATACTTCGGTTGCATGAAGGCG Isoform 3 and 6 [SEQ ID No: 24] MQSREEEWFHQVRRVMTILFLTMVISYFGCMKA [SEQ ID No: 25] ATGCAGAGCCGGGAAGAGGAATGGTTCCACCAGGTGAGAAGAGTGATGAC CATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG Isoform 5 [SEQ ID No: 26] MLCAISLCARVRKLRSAGRCGKFHQVRRVMTILFLTMVISYFGCMKA [SEQ ID No: 27] ATGCTCTGTGCGATTTCATTGTGTGCTCGCGTTCGCAAGCTCCGTAGTGC AGGAAGGTGCGGGAAGTTCCACCAGGTGAGAAGAGTGATGACCATCCTTT TCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG Isoform 4 [SEQ ID No: 28] MCGATSFLHECTRLILVTTQNAEFLQKGLQVHTCFGVYPHASVWHDCASQ KKGCAVYLHVSVEFNKLIPENGFIKFHQVRRVMTILFLTMVISYFGCMKA [SEQ ID No: 29] ATGTGTGGAGCCACCAGTTTTCTCCATGAGTGCACAAGGTTAATCCTTGT TACTACTCAGAATGCTGAGTTTCTACAGAAAGGGTTGCAGGTCCACACAT GTTTTGGCGTCTACCCACACGCTTCTGTATGGCATGACTGTGCATCCCAG AAGAAGGGCTGTGCTGTGTACCTCCACGTTTCAGTGGAATTTAACAAACT GATCCCTGAAAATGGTTTCATAAAGTTCCACCAGGTGAGAAGAGTGATGA CCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG

Accordingly, in preferred embodiments, the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide referred to herein as any one of SEQ ID NO. 23, 25, 27 or 29. Preferably, the signal peptide comprises an amino acid sequence referred to herein as any one of SEQ ID NO. 22, 24, 26 or 28.

The inventors have also created various embodiments of novel signal peptides for the agonist, preferably BDNF. These signal peptides increase the level of basicity of the N-terminal section (with added lysine (K) and arginine (R) residues) and the proceeding hydrophobic region (with additions of leucine (L) residues), which increase secretion of BDNF compared to levels observed with the wild-type canonical signal sequence.

a) QTA003P (IL-2 signal) [SEQ ID No: 30] MYRMQLLSCIALSLALVTNS [SEQ ID No: 31] ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGT CACAAACAGT b) QTA004P [SEQ ID No: 32] MKRRVMIILFLTMVISYFGCMK [SEQ ID No: 33] ATGAAAAGAAGAGTGATGATCATCCTTTTCCTTACTATGGTTATTTCATA CTTCGGTTGCATGAAGAGCG c) QTA009P (modified IL-2) [SEQ ID No: 34] MRRMQLLLLIALSLALVTNS [SEQ ID No: 35] ATGAGGAGGATGCAACTCCTGCTCCTGATTGCACTAAGTCTTGCACTTGT CACAAACAGT d) QTA010P [SEQ ID No: 36] MRRMQLLLLTMVISYFGCMKA [SEQ ID No: 37] ATGAGGAGGATGCAACTCCTGCTCCTGACTATGGTTATTTCATACTTCGG TTGCATGAAGGCG e) QTA0012P [SEQ ID No: 38] MRILLLTMVISYFGCMKA [SEQ ID No: 39] ATGAGAATCCTTCTTCTTACTATGGTTATTTCATACTTCGGTTGCATGAA GGCG f) QTA0013P [SEQ ID No: 40] MRRILFLTMVISYFGCMKA [SEQ ID No: 41] ATGAGAAGAATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCAT GAAGGCG g) QTA0014P [SEQ ID No: 42] MRRFLFLLVISYFGCMKA [SEQ ID No: 43] ATGAGGAGGTTCCTTTTCCTTCTTGTTATTTCATACTTCGGTTGCATGAA GGCG i) QTA0015P [SEQ ID No: 44] MRRFLFLLYFGCMKA [SEQ ID No: 45] ATGAGGAGGTTCCTTTTCCTTCTTTACTTCGGTTGCATGAAGGCG

FIG. 6 shows nucleotide and amino acid sequences for further preferred embodiments of signal peptide used in the construct of the invention to boost secretion of the agonist, preferably BDNF. The second residue in the signal peptide is threonine (T) which is preferably replaced by one or more basic residue, such as lysine (K) or arginine (R). The next stretch of residues in the signal peptide including isoleucine (I), leucine (L), phenylalanine (F) and Leucine (L) is preferably replaced by one or more hydrophobic residues.

Accordingly, in preferred embodiments, the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide referred to herein as any one of SEQ ID NO. 31, 33, 35, 37, 39, 41, 43, 45, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, lot or 103. Preferably, the signal peptide comprises an amino acid sequence referred to herein as any one of SEQ ID NO. 30, 32, 34, 36, 38, 40, 42, 44, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102.

Accordingly, it will be appreciated that the inventors have modified the BDNF gene sequence by removal of the pro-sequence, which also has never been achieved before, with the result of generated properly folded mature BDNF, combined with the introduction of completely novel signal peptides, which significantly boost BDNF production and release above that ever achieved with the endogenous sequence.

Preferably, the genetic construct comprises left and/or right Inverted Terminal Repeat sequences (ITRs). Preferably, each ITR is disposed at the 5′ and/or 3′ end of the construct. An ITR can be specific to a virus (e.g. AAV or lentivirus) serotype, and can be any sequence, so long as it forms a hairpin loop in its secondary structure.

The DNA sequence of one embodiment (left ITR from a commercially available AAV plasmid) of the ITR is represented herein as SEQ ID No: 46, as follows:

[SEQ ID NO: 46] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCG GGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAACTCCATCACTAGGGGTTCCT

The DNA sequence of another embodiment (right ITR from a commercially available AAV plasmid) of the ITR is represented herein as SEQ ID No: 47, as follows:

[SEQ ID NO: 47] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCG CTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG From the foregoing, the skilled person will appreciate the nucleotide sequence of an embodiment of the construct of the first aspect, as well as the amino acid sequence of the encoded transgene. However, for the avoidance of doubt, the coding sequence of codon optimised 2940 bp sequence for murine TrkB receptor-viral-2A peptide-mBDNF contained within the plasmid QTA020P (and the vector QTA020V), is referred to here as SEQ ID No: 107, as follows:

[SEQ ID No: 107] ATGAGCCCATGGCTGAAGTGGCACGGACCAGCAATGGCAAGACTGTGGGG CCTGTGCCTGCTGGTGCTGGGCTTCTGGAGAGCCAGCCTGGCCTGTCCAA CCTCCTGCAAGTGTAGCTCCGCCAGGATCTGGTGCACAGAGCCTTCTCCA GGCATCGTGGCCTTTCCCCGCCTGGAGCCTAACAGCGTGGATCCCGAGAA TATCACCGAGATCCTGATCGCCAACCAGAAGCGGCTGGAGATCATCAATG AGGACGATGTGGAGGCCTACGTGGGCCTGAGAAACCTGACAATCGTGGAC TCCGGCCTGAAGTTCGTGGCCTATAAGGCCTTTCTGAAGAACTCTAATCT GAGGCACATCAACTTCACCCGCAATAAGCTGACATCTCTGAGCCGGAGAC ACTTTCGGCACCTGGATCTGTCCGACCTGATCCTGACCGGCAATCCATTC ACATGCTCTTGTGACATCATGTGGCTGAAGACCCTGCAGGAGACAAAGTC TAGCCCCGATACCCAGGACCTGTACTGTCTGAACGAGTCCTCTAAGAATA TGCCTCTGGCCAACCTGCAGATCCCTAATTGTGGACTGCCAAGCGCCCGG CTGGCCGCACCTAACCTGACAGTGGAGGAGGGCAAGTCCGTGACACTGTC CTGTTCTGTGGGCGGCGATCCCCTGCCTACCCTGTATTGGGACGTGGGCA ACCTGGTGTCTAAGCACATGAATGAGACCTCCCACACACAGGGCTCTCTG AGAATCACAAATATCAGCTCCGACGATAGCGGCAAGCAGATCTCTTGCGT GGCAGAGAACCTGGTGGGAGAGGATCAGGACAGCGTGAATCTGACCGTGC ACTTCGCCCCCACCATCACATTTCTGGAGTCTCCTACCAGCGATCACCAC TGGTGCATCCCCTTCACAGTGCGGGGAAACCCAAAGCCCGCCCTGCAGTG GTTTTACAACGGCGCCATCCTGAATGAGTCCAAGTATATCTGTACCAAGA TCCACGTGACCAACCACACAGAGTACCACGGCTGCCTGCAGCTGGATAAT CCCACCCACATGAACAATGGCGACTACACACTGATGGCCAAGAACGAGTA TGGCAAGGACGAGAGGCAGATCAGCGCCCACTTCATGGGCCGCCCTGGAG TGGATTATGAGACCAACCCTAATTACCCAGAGGTGCTGTATGAGGACTGG ACCACACCTACCGATATCGGCGACACCACAAACAAGTCTAATGAGATCCC AAGCACAGATGTGGCCGACCAGTCTAACAGGGAGCACCTGAGCGTGTACG CAGTGGTGGTCATCGCCTCCGTGGTGGGCTTCTGCCTGCTGGTCATGCTG CTGCTGCTGAAGCTGGCCCGCCACTCTAAGTTTGGCATGAAGGGCCCAGC CTCCGTGATCTCTAATGACGATGACAGCGCCAGCCCCCTGCACCACATCA GCAACGGCTCCAATACCCCTTCTAGCTCCGAGGGCGGCCCAGATGCCGTG ATCATCGGCATGACAAAGATCCCCGTGATCGAGAACCCTCAGTACTTCGG CATCACCAATTCCCAGCTGAAGCCTGACACATTTGTGCAGCACATCAAGC GGCACAACATCGTGCTGAAGAGGGAACTGGGAGAGGGAGCCTTCGGCAAG GTGTTTCTGGCCGAGTGCTATAACCTGTGCCCAGAGCAGGATAAGATCCT GGTGGCCGTGAAGACCCTGAAGGATGCCAGCGACAACGCCCGGAAGGACT TCCACAGAGAGGCCGAGCTGCTGACAAATCTGCAGCACGAGCACATCGTG AAGTTTTACGGCGTGTGCGTGGAGGGCGACCCTCTGATCATGGTGTTCGA GTATATGAAGCACGGCGATCTGAACAAGTTTCTGAGAGCACACGGACCAG ATGCCGTGCTGATGGCAGAGGGAAATCCCCCTACCGAGCTGACACAGTCT CAGATGCTGCACATTGCACAGCAGATTGCAGCAGGAATGGTGTACCTGGC CAGCCAGCACTTCGTGCACAGGGATCTGGCAACCAGAAACTGCCTGGTGG GAGAGAATCTGCTGGTGAAGATCGGCGACTTTGGCATGTCCCGGGACGTG TACTCTACCGACTACTATAGAGTGGGCGGCCACACAATGCTGCCCATCAG GTGGATGCCACCCGAGAGCATCATGTATCGCAAGTTCACCACAGAGTCTG ACGTGTGGAGCCTGGGCGTGGTGCTGTGGGAGATCTTTACCTACGGCAAG CAGCCTTGGTATCAGCTGTCCAACAATGAAGTGATCGAGTGTATTACACA GGGACGCGTGCTGCAGAGGCCACGCACATGCCCCCAGGAGGTGTACGAGC TGATGCTGGGCTGTTGGCAGCGGGAGCCACACACCAGAAAGAACATCAAG AGCATCCACACACTGCTGCAGAATCTGGCCAAGGCCTCCCCCGTGTATCT GGACATCCTGGGCAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTG GAGACGTGGAGGAGAACCCTGGACCTATGAGAATCCTTCTTCTTACTATG GTTATTTCATACTTCGGTTGCATGAAGGCGCACTCCGACCCTGCCCGCCG TGGGGAGCTGAGCGTGTGTGACAGTATTAGCGAGTGGGTCACAGCGGCAG ATAAAAAGACTGCAGTGGACATGTCTGGCGGGACGGTCACAGTCCTAGAG AAAGTCCCGGTATCCAAAGGCCAACTGAAGCAGTATTTCTACGAGACCAA GTGTAATCCCATGGGTTACACCAAGGAAGGCTGCAGGGGCATAGACAAAA GGCACTGGAACTCGCAATGCCGAACTACCCAATCGTATGTTCGGGCCCTT ACTATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACAC TTCCTGTGTATGTACACTGACCATTAAAAGGGGAAGATAG

The coding sequence of codon optimised 2943 bp sequence for human TrkB receptor-viral-2A peptide-mBDNF contained within the plasmid QTA029P (and the vector QTA029V), is referred to here as SEQ ID No: 108, as follows:

[SEQ ID No: 108] ATGTCATCTTGGATCCGCTGGCACGGGCCAGCGATGGCCCGATTGTGGGG CTTCTGCTGGCTTGTTGTAGGCTTCTGGCGCGCGGCGTTCGCGTGTCCGA CCTCTTGCAAATGCTCAGCAAGCCGAATTTGGTGCTCAGACCCTAGTCCA GGAATTGTTGCATTCCCCCGACTGGAACCAAACTCCGTCGACCCGGAGAA TATAACTGAGATATTTATTGCAAATCAAAAACGCCTTGAAATCATTAACG AGGATGACGTGGAGGCCTACGTTGGTTTGAGAAATCTTACTATTGTCGAC TCCGGACTTAAATTTGTAGCTCATAAAGCCTTCCTGAAGAACTCTAATCT GCAGCACATTAATTTCACGAGAAATAAGCTGACCAGCTTGTCCCGGAAGC ATTTCCGCCATCTCGACCTGAGCGAGCTCATACTGGTCGGAAACCCATTT ACGTGCTCCTGTGACATCATGTGGATCAAAACTCTGCAAGAGGCGAAAAG TAGTCCGGATACCCAAGACCTTTACTGTCTTAATGAAAGCTCAAAAAATA TCCCGCTGGCCAACCTGCAGATACCGAACTGCGGACTTCCTAGTGCGAAT TTGGCTGCCCCAAATCTTACCGTCGAAGAAGGCAAATCAATCACGCTTTC TTGTTCTGTAGCTGGAGATCCAGTGCCTAATATGTATTGGGACGTGGGTA ACCTCGTCTCAAAACATATGAACGAAACGAGCCACACCCAGGGCTCTTTG CGGATAACAAACATCTCCTCTGATGATTCTGGAAAGCAAATCAGTTGCGT AGCTGAAAATCTGGTTGGCGAAGATCAAGATTCAGTCAATCTGACAGTCC ATTTCGCCCCAACGATCACCTTTCTGGAGAGCCCAACTAGCGATCACCAC TGGTGTATTCCGTTTACGGTAAAAGGAAATCCAAAACCTGCACTCCAATG GTTTTATAATGGAGCCATCTTGAATGAAAGCAAATATATCTGTACTAAAA TCCATGTGACGAATCACACCGAGTATCACGGGTGTCTTCAATTGGATAAT CCAACCCATATGAATAATGGTGATTATACTTTGATAGCGAAGAACGAATA CGGCAAAGACGAAAAGCAAATATCCGCACATTTCATGGGTTGGCCTGGCA TCGACGACGGTGCGAACCCGAACTACCCAGATGTTATTTACGAGGATTAT GGGACTGCGGCAAACGACATTGGCGACACCACAAACCGAAGCAACGAGAT ACCAAGTACTGACGTCACTGACAAAACGGGTCGAGAGCATTTGTCTGTTT ACGCCGTTGTTGTTATCGCCTCAGTTGTCGGATTTTGCCTGTTGGTCATG CTTTTCCTCCTGAAGCTCGCGCGACATTCCAAGTTTGGCATGAAGGGGCC AGCAAGTGTTATATCCAATGATGATGATAGCGCTTCTCCATTGCACCACA TAAGTAACGGCTCAAACACGCCGTCATCTAGTGAAGGTGGACCAGACGCG GTCATTATAGGGATGACTAAAATTCCCGTAATCGAAAACCCTCAGTACTT CGGCATAACCAACAGTCAGCTTAAACCCGATACTTTCGTGCAGCACATCA AAAGGCACAACATAGTCCTCAAGCGCGAACTCGGGGAGGGAGCCTTCGGA AAGGTCTTTCTTGCTGAGTGCTATAATTTGTGTCCTGAGCAGGATAAAAT TCTTGTGGCTGTAAAAACTCTCAAAGATGCTTCCGACAACGCACGGAAGG ATTTTCATCGGGAGGCCGAACTGTTGACGAATTTGCAGCACGAGCATATA GTAAAGTTCTACGGGGTATGTGTTGAGGGGGACCCGTTGATTATGGTCTT CGAGTATATGAAGCACGGGGACCTGAACAAATTTTTGCGCGCCCATGGGC CTGATGCCGTCCTTATGGCAGAAGGGAACCCTCCAACAGAACTCACCCAG AGTCAGATGTTGCACATAGCGCAACAGATCGCGGCCGGCATGGTTTACCT GGCCAGTCAACACTTCGTGCATAGAGATCTTGCCACTCGCAACTGTTTGG TCGGGGAGAACCTTCTGGTTAAGATTGGTGACTTTGGTATGTCACGAGAT GTGTATTCCACTGACTATTACAGAGTTGGGGGTCATACAATGCTTCCTAT TCGGTGGATGCCCCCCGAATCCATCATGTACAGAAAGTTCACGACAGAGA GTGATGTTTGGAGTCTCGGCGTGGTGCTCTGGGAAATTTTCACATACGGA AAGCAGCCGTGGTATCAACTTAGCAACAATGAGGTGATAGAGTGTATTAC ACAGGGTCGGGTGTTGCAGCGCCCTCGAACGTGCCCACAAGAAGTATATG AACTTATGCTCGGGTGCTGGCAAAGAGAACCACATATGAGAAAAAATATC AAGGGGATACATACATTGCTTCAGAACTTGGCCAAGGCATCACCCGTCTA CCTCGATATACTGGGCAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGG CTGGAGACGTGGAGGAGAACCCTGGACCTATGAGAATCCTTCTTCTTACT ATGGTTATTTCATACTTCGGTTGCATGAAGGCGCACTCCGACCCTGCCCG CCGTGGGGAGCTGAGCGTGTGTGACAGTATTAGCGAGTGGGTCACAGCGG CAGATAAAAAGACTGCAGTGGACATGTCTGGCGGGACGGTCACAGTCCTA GAGAAAGTCCCGGTATCCAAAGGCCAACTGAAGCAGTATTTCTACGAGAC CAAGTGTAATCCCATGGGTTACACCAAGGAAGGCTGCAGGGGCATAGACA AAAGGCACTGGAACTCGCAATGCCGAACTACCCAATCGTATGTTCGGGCC CTTACTATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGA CACTTCCTGTGTATGTACACTGACCATTAAAAGGGGAAGATAG

Hence, in a most preferred embodiment, the construct comprises a nucleotide sequence substantially as set out in SEQ ID No: 107 or 108, or a fragment or variant thereof.

The inventors have created a series of recombinant expression vectors comprising the construct of the invention.

Thus, according to a second aspect, there is provided a recombinant vector comprising the genetic construct according to the first aspect.

The recombinant vector may be a recombinant AAV (rAAV) vector. The rAAV may be a naturally occurring vector or a vector with a hybrid AAV serotype. The rAAV may be AAV-1, AAV-2, AAV-3A, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, and AAV-11. Preferably, the rAAV is rAAV serotype-2.

Advantageously, recombinant AAV2 evokes a minimal immune response in host organisms and mediates long-term transgene expression that can persist in the retina for at least one year after vector administration.

The term “recombinant AAV (rAAV) vector” as used herein means a recombinant AAV-derived nucleic acid containing at least one terminal repeat sequence.

Preferred embodiments of the vector are shown in FIGS. 2-5.

The constructs and expression vectors described herein can be used to treat optic nerve disorders and a cochlear disorders, and more generally to promote nerve regeneration and survival.

Hence, according to a third aspect, there is provided the genetic construct according to the first aspect, or the recombinant vector according to the second aspect, for use as a medicament or in therapy.

According to a fourth aspect, there is provided the genetic construct according to the first aspect, or the recombinant vector according to the second aspect, for use in treating, preventing or ameliorating an optic nerve disorder or a cochlear disorder, or for promoting nerve regeneration and/or survival.

According to a fifth aspect, there is provided a method of treating, preventing or ameliorating an optic nerve disorder or a cochlear disorder in a subject, or for promoting nerve regeneration and/or survival in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the genetic construct according to the first aspect, or the recombinant vector according to the second aspect.

Preferably, the genetic construct or the recombinant vector according to invention are used in a gene therapy technique. The agonist encoded by the construct or vector activate the TrkB also encoded by the construct/vector to thereby promote survival of retinal ganglion cells (RGCs) or cochlear cells.

In one embodiment, the optic nerve disorder that is treated may be glaucoma, or any other pathophysiological condition which may result in loss of RGCs, such as trauma to the head or face or vascular insults, for example partial or complete loss in blood supply to the ocular structures or regions of the brain which receive input from the optic nerve.

In addition, the construct may also be used to support replacement of RGCs through introduction of untransformed or transformed stem cell into the eye or regions associated with vision in patients.

In one embodiment, the cochlear disorder which is treated may be hearing loss or deafness. The constructs and vectors of the invention significantly enhance cochlear cell sensitivity to TrkB receptor agonists due to a localised increase in both the TrkB receptor and the agonist of the receptor. The cochlear cells may be hair cells or neuronal spiral ganglion cells which send auditory signals via their axons from the ear to the brainstem. The hair cells may be inner ear hair cells or outer ear hair cells [42, 43, 44].

In another embodiment, the constructs and vectors may be used to promote nerve regeneration and/or survival.

It will be appreciated that the genetic construct according to the first aspect, or the recombinant vector according to the second aspect may be used in a medicament, which may be used as a monotherapy (i.e. use of the genetic construct according to the first aspect or the vector according to the second aspect of the invention), for treating, ameliorating, or preventing an optic nerve disorder or a cochlear disorder, or for promoting nerve regeneration and/or survival. Alternatively, the genetic construct or the recombinant vector according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing an optic nerve disorder or a cochlear disorder, or for promoting nerve regeneration and/or survival.

The genetic construct according or the recombinant vector according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given.

The genetic construct or the recombinant vector according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with the genetic construct or the recombinant vector is required and which would normally require frequent administration (e.g. at least daily injection).

In a preferred embodiment, medicaments according to the invention may be administered to a subject by injection into the blood stream, a nerve or directly into a site requiring treatment. For example, the medicament may be injected at least adjacent the retina or ear. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the genetic construct or the recombinant vector that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the genetic construct or the recombinant vector and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the cyclic polypeptide within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular genetic construct or the recombinant vector in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the optic nerve disorder or the cochlear disorder. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10 mg/kg of body weight, or between 0.01 μg/kg of body weight and 1 mg/kg of body weight, of the cyclic polypeptide according to the invention may be used for treating, ameliorating, or preventing an optic nerve disorder or a cochlear disorder, depending upon the genetic construct or recombinant vector used.

The genetic construct or the recombinant vector may be administered before, during or after onset of the optic nerve or cochlear disorder. Daily doses may be given as a single administration (e.g. a single daily injection or inhalation of a nasal spray).

Alternatively, the genetic construct or the recombinant vector may require administration twice or more times during a day. As an example, the genetic construct or the recombinant vector may be administered as two (or more depending upon the severity of the optic nerve or cochlear disorder being treated) daily doses of between 0.07 μg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of the genetic construct or the recombinant vector according to the invention to a patient without the need to administer repeated doses.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the genetic construct or the recombinant vector according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). The inventors believe that they are the first to suggest a genetic construct encoding promoter operably linked to coding sequences of a TrkB receptor and a TrkB receptor agonist.

According to a sixth aspect, there is provided a pharmaceutical composition comprising the genetic construct according to the first aspect, or the recombinant vector according to the second aspect, and a pharmaceutically acceptable vehicle.

According to a seventh aspect, there is provided a method of preparing the pharmaceutical composition according to the sixth aspect, the method comprising contacting the genetic construct according to the first aspect, or the recombinant vector according to the second aspect, with a pharmaceutically acceptable vehicle.

A “subject” may be a vertebrate, mammal, or domestic animal. Hence, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.

A “therapeutically effective amount” of the genetic construct, the recombinant vector or the pharmaceutical composition is any amount which, when administered to a subject, is the amount of the aforementioned that is needed to treat glaucoma, deafness or produce the desired effect, such as promoting nerve regeneration and/or survival.

For example, the therapeutically effective amount of the genetic construct, the recombinant vector or the pharmaceutical composition used may be from about 0.01 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg. It is preferred that the amount of the genetic construct, the recombinant vector or the pharmaceutical composition is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. the genetic construct or recombinant vector according to the invention) may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The genetic construct or the recombinant vector according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The genetic construct or the recombinant vector may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The genetic construct, the recombinant vector and the pharmaceutical composition of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The genetic construct, the recombinant vector or the pharmaceutical composition according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms “substantially the amino acid/nucleotide/peptide sequence”, “variant” and “fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:1-108, and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:—(i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity. For protein alignments: Gap Open Penalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:—Sequence Identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3× sodium chloride/sodium citrate (SSC) at approximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in, for example, SEQ ID Nos: 3 and 5.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

According to another aspect, there is provided a genetic construct comprising a promoter operably linked to a first coding sequence, which encodes the tyrosine kinase receptor B (TrkB), and a second coding sequence, which encodes an agonist of the TrkB receptor for activating TrkB to thereby promote survival of retinal ganglion cells (RGCs), nerve cells or cochlear cells.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figure, in which:—

FIG. 1 is schematic of one embodiment of a genetic construct according to the invention;

FIG. 2 is a schematic drawing of a first embodiment of a recombinant vector according to the invention known as “Plasmid QTA001PA” containing canonical signal sequence (blue) plus proBDNF (red) and mBDNF (black). It also includes an -IRES-GFP- sequence (cyan and purple);

FIG. 3 is a schematic drawing of a second embodiment of the recombinant vector according to the invention known as “Plasmid QTA002P” with no proBDNF (but produces only mBDNF) and same signal sequence (blue) as QTA001PA. It also includes an -IRES-GFP- sequence (cyan and purple);

FIG. 4 is a schematic drawing of a third embodiment of the recombinant vector according to the invention known as of “Plasmid QTA003P” with no proBDNF (but produces only mBDNF) and IL-2 signal sequence (blue). It also includes an -IRES-GFP- sequence (cyan and purple);

FIG. 5 is a schematic drawing of a fourth embodiment of a recombinant vector according to the invention known as “Plasmid QTA004P” with no proBDNF (but produces only mBDNF) and a novel signal sequence (blue). It also includes an -IRES-GFP- sequence (cyan and purple);

FIG. 6 shows nucleotide and amino acid sequences for different embodiments of signal peptide used in the construct of the invention. The second residue is threonine (t) which can be replaced by one or more basic residue, such as lysine (K) or arginine (R). The next stretch of residues including isoleucine (I), leucine (L), phenylalanine (F) and Leucine (L) can be replaced by one or more hydrophobic residues;

FIG. 7 shows release of BDNF from HEK293 cells using a specific ELISA at 24 hours following transduction of a plasmid (4 μg DNA/well) containing genes coding for mBDNF with differing signal peptide sequences and without the coding sequence for the extended proBDNF component (Data shown as mean±SEM for n=4);

FIG. 8 shows Western blotting results of cellular concentrations of BDNF-immunoreactive material (arbitrary units) in HEK293 cell lysates 24 hours after plasmid transduction (Data shown as mean±SEM for n=4);

FIG. 9 shows BDNF-immunoreactivity in Western blots of cell lysates showing two molecular weight bands (32 kDa and 14 kDa) when cells were transduced with QTA001PA, versus only a single 14 kDa band with QTA002P, QTA003P and QTA004P transduction;

FIG. 10 shows proBDNF concentrations in the HEK293 incubation medium as measured using a specific ELISA 24 hours after plasmid transduction using a selective proBDNF ELISA (Data shown as mean±SEM for n=4);

FIG. 11 shows BDNF expression in HEK293 cell lysate by plasmids QTA002P (endogenous canonical signal peptide sequence), and QTA009P to QTA013P. Data is shown as mean+S.E.M. *** P<0.01 as compared to QTA002P;

FIG. 12 shows BDNF expression in HEK293 cell incubation medium by plasmids QTA002P (endogenous canonical signal peptide sequence), and QTA009P to QTA013P. Data is shown as mean+S.E.M. *** P<0.01 as compared to QTA002P;

FIG. 13 shows Western Blots from HEK293 cells 24 hours after they were transduced with plasmids QTA015P (expressing BDNF and eGFP separated by an IRES spacer), QTA021P (expressing BDNF followed by eGFP separated by a functional viral-2A peptide sequence), QTA022P (expressing BDNF followed by eGFP separated by a non-functional viral-2A peptide sequence) and QTA023P (expressing eGFP followed by coding for BDNF separated by a functional viral-2A peptide sequence). Data shown as BDNF-immunoreactivity (A), eGFP-immunoreactivity (B) and the amount of BDNF released from the HEK293 cells into the incubation medium (C). Data is shown as mean+S.E.M of the density in the bands;

FIG. 14A shows Western blot of HEK293 cell homogenates 48 hours after transfection with the QTA020V vector and showing efficient processing of the large precursor coding region which includes the TrkB receptor and BDNF separated by the viral-2A peptide sequence. FIGS. 14B and 14C show that the transgene proteins produced after vial-2A peptide cleavage have been transported to the correct intracellular compartments in HEK293 cells after processing (TrkB receptors to the cell surface and BDNF to storage vesicles prior to release);

FIG. 15A shows TrkB receptor expression and FIG. 15B shows BDNF expression in mouse retinal homogenate for the rAAV2 vector, QTA020V. Data is shown as mean+S.E.M of the density in the Western blot of mouse retina homogenates. ** P<0.01 as compared to naïve (un-injected animals);

FIG. 16 shows expression of TrkB (A) and BDNF (B) transgenes in mouse retinal ganglion cell layer as shown by immunocytochemistry following injection of QTA020V, a rAAV2 vector containing the coding for the TrkB receptor and BDNF, separated by the viral-2A peptide sequence; and

FIG. 17 shows retinal ganglion cell (RGC) survival following optic nerve crush (ONC) in the mouse versus control animals treated with rAAV2-CAG-eGFP vector. Data shown as mean+S.E.M. for average numbers of retinal ganglion cells throughout the retina per animal as counted by Brn3A-positive cells in retinal flat-mounts. ***P<0.001, *P<0.05 as compared to controls.

EXAMPLES

Methods and Materials

Molecular Cloning and Plasmid Constructs

Codon optimisation of DNA sequences was performed using the on-line tool (http://www.idtdna.com/CodonOpt) and DNA blocks were synthesised by Integrated DNA technologies, Inc. (IDT; 9180 N. McCormick Boulevard, Skokie, Ill. 60076-2920, USA) or GenScript (860 Centennial Ave, Piscataway, N.J. 08854, USA). Cloning to make the master plasmid QTA001PA and subsequent plasmids were performed using standard molecular biology and cloning techniques.

Plasmid Scale Up and Purification

DNA Plasmids were scaled up in SURE competent cells (Agilent Technologies; cat. #200238) overnight to provide 2.29 μg/μl plasmid following maxi-prep purification. The remaining plasmids were scaled up to 500 μg scale and transduction quality with minimal endotoxin presence.

HEK293 Culture and Cell Transduction with Plasmid DNA

HEK293 cells (400,000 cells) were cultured in poly-L-lysine (10 ug/mL, Sigma-Aldrich; cat. #P1274) coated 6 well plates in 1.5 mL Dulbecco's minimum essential medium (DMEM) containing 10% foetal bovine serum (FBS), 1% penicillin and 1% streptomycin (1% Pen/Strep) until 80% confluent. The medium was then exchanged for 2 mL DMEM (no additives). Two to three hours later, an additional 0.5 ml transfection medium containing 4 μg plasmid DNA plus 10 μL lipofectamine (4 μL/mL; Thermo Fisher Scientific; cat. #12566014) was added to each well resulting in an overall volume of 2.5 ml throughout the transfection period and for supernatant collection.

BDNF Measurement by ELISA

The amount of BDNF secreted from HEK293 cells was measured in cell culture medium 24 hours after transfection. Medium was centrifuged, to remove debris, and measured using a commercial Human BDNF ELISA kit (Sigma-Aldrich, product #RAB0026). BDNF concentration was determined by comparing samples to freshly made BDNF standards.

Western Blotting for BDNF and TrkB Receptors

The amount of BDNF and TrkB-immunoreactivity within the HEK293 cells was measured by removing the DMEM incubation medium, washing the cells in cold phosphate buffered saline and the addition of 350 μL freshly prepared lysis buffer to the wells (10 ml Lysis-M reagent+1 tablet of complete Mini Protease Inhibitor Cocktail, Roche; cat. #04719964001, +100 μl Halt phosphatase inhibitor cocktail (100×), Thermo Scientific; cat. #78428). After cell homogenisation, the protein suspension was quantified using the BCA assay (Pierce BCA protein assay kit, Thermo Scientific; cat. #23227). Between 6 μg and 15 μg HEK293 cell lysate protein/lane were run down a Bis-Tris gel (12% NuPAGE Novex; cat. #NP0342BOX, Thermo Scientific) and examined by Western blotting using the primary rabbit polyclonal anti-BDNF antibodies (Santa Cruz Biotechnology Inc; product #sc-546; at 1:500 dilution), rabbit polyclonal anti-TrkB antibodies (Abcam; cat. #ab33655, used at 1:2000 dilution) or eGFP antibodies (Abcam product #ab-290 used at 1:500) which were incubated overnight. Primary antibodies were visualised with HRP conjugated anti-rabbit antibodies (Vector Laboratories; cat. #PI-1000, at 1:8000) and signal detection using ECL Prime (Amersham, GE Healthcare, UK) and an Alliance Western blot imaging system (UVItec Ltd, Cambridge, UK). For Western blots of mouse retina, eyes from vector-treated animals were homogenized in 500 μL freshly prepared lysis buffer (10 ml Lysis-M reagent+1 tablet of cOmplete Mini Protease Inhibitor Cocktail, Roche product #04719964001+100 μl Halt phosphatase inhibitor cocktail (100×), Thermo Scientific product #78428). Tissue was disrupted for 1 minute (Qiagen, TissueRuptor product #9001273) and then kept on ice for an additional 15 minutes. The protein was then analysed by Western blotting as described above.

Immunocytochemistry

HEK293 cells (70,000) were seeded on 13 mm, poly-L-lysine coated coverslips within 4 well plates and incubated in DMEM containing 10% FBS and 1% Pen/Strep in 0.5 ml medium. Once the cells had grown to 80% confluence, the medium was exchanged for 0.4 ml DMEM (no additives) for 2-3 hours then an additional 0.1 mL transfection medium (0.8 μg plasmid DNA+2 μl lipofectamine) was added so that the final volume reached 0.5 ml. Coverslips were washed twice in PBS and fixed for 30 min in 4% paraformaldehyde in 1M phosphate buffered saline (PBS) at room temperature. After three more washes in PBS, cells were blocked and permeabilized by incubation in 5% normal goat serum (NGS), 3% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS for 60 minutes at room temperature. Cells were then incubated overnight at 4° C. with commercial rabbit polyclonal antibodies for BDNF (Santa Cruz Biotechnology Inc; product #sc-546; at 1:300 dilution) or TrkB (Abcam product #ab33655, diluted 1:500) diluted in blocking solution. Staining was revealed using secondary anti-rabbit antibodies conjugated to alexa fluor 647 (Invitrogen, product #A21248 at 1:1000) for 2 hours at room temperature. Cell nuclei were also counterstained with 1 μg/ml DAPI (Thermo Scientific, product #D1306 at 1:8000). Cells were further washed three times before being mounted with FluorSave™ reagent (Calbiochem/EMD Chemicals Inc., Gibbstown, N.J., USA) prior to imaging. Imaging was carried out using a 20× objective and a Leica DM6000 epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) or a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63× oil objective using a 3× digital zoom and 0.5-0.8 sequential scanning z-step interval.

For immunocytochemistry of retinal structures from control or vector treated animals, carefully dissected eyes were fixed in 4% paraformaldehyde/0.1% PBS (pH 7.4) overnight and dehydrated in 30% sucrose/0.1% PBS at 4° C. (24 hours). Eyes were then embedded in silicon moulds containing optimal cutting temperature compound (OCT) (Sakura Finetek, Zoeterwoude, Netherlands) and frozen on dry ice. 13 μm sections through the dorsal-ventral/superior-inferior axis of the retina were collected onto superfrost plus slides (VWR product #631-0108), using a Bright OTF 5000 cryostat (Bright Instruments, Huntingdon, UK). Slides were washed three times in PBS, and permeabilized in 5% normal goat serum (NGS), 3% bovine serum albumin (BSA) and 0.3% Triton X-100 in PBS for 60 minutes at room temperature. Slides were then incubated overnight at 4° C. with commercial rabbit polyclonal antibodies for BDNF (Santa Cruz Biotechnology Inc product #sc-546 1:300) or TrkB (Abcam product #ab33655 1:500), diluted in blocking solution. Staining was revealed using secondary anti-rabbit antibodies conjugated to alexa fluor 647 (Invitrogen, product #A21248 at 1:1000) for 2 hours at room temperature. Retinal cell nuclei were also counterstained with 1 μg/mL DAPI (Thermo Scientific, product #D1306 at 1:8000). Slides were further washed three times before being mounted with FluorSave™ reagent (Calbiochem/EMD Chemicals Inc., Gibbstown, N.J., USA) prior to imaging. Imaging was carried out using a 20× objective and a Leica DM6000 epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) or a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63× oil objective using a 3× digital zoom and 0.5-0.8 sequential scanning z-step interval.

Intravitreal Injections

Following a 7-10 day acclimatisation period, mice were randomised into various study groups. They were then anaesthetized with intraperitoneal injection of ketamine (50 mg/kg) and xylazine (5 g/kg). Topical 1% tetracaine eye drops were administered on Day 1 of the study. Pupillary dilation was achieved using 1% tropicamide eye drops. Using an operating microscope, a partial-thickness scleral pilot hole was made with a 30-gauge needle to facilitate penetration of the underlying sclera, choroid, and retina by a fine metal micropipette with a tip diameter of 30 μm and a tip length of 2.5 mm. The micropipette was then connected to a 10 μl glass syringe (Hamilton Co., Reno, Nev.) prior drawing up 2 μl of vector suspensions into the pipette depending on the group. Care was taken to avoid penetration of the lens or damage to the vortex veins during intravitreal injection. The injection site was aimed approximately 3 mm posterior to the supero-temporal limbus. Injections were given slowly over 1 minute to allow diffusion of vector suspension. The right eye was left untouched and served as an internal contralateral control.

Optic Nerve Crush (ONC)

Three weeks (21 days) after vector administration, the mice were subject to the ONC procedure, left untreated or sham-crushed. Under a binocular operating scope, a small incision was made with spring scissors in the conjunctiva beginning inferior to the globe and around the eye temporally. This exposed the posterior aspect of the globe, allowing visualization of the optic nerve. The exposed optic nerve was grasped approximately 1-3 mm from the globe with cross-action forceps (Dumont #N7 cat. #RS-5027; Roboz) for 10 s, with the only pressure from the self-clamping action to press on the nerve. After 10 s the optic nerve was released, the forceps are removed and the eye rotates back into place. 7 days after ONC, animals were culled. Both eyes from each group were fixed by placing the organ in 4% paraformaldehyde/0.1% PBS (pH 7.4) overnight. Retinal flat-mounts were then prepared following dissection of the posterior eye structure from the cornea and removal of the lens. The retinal flat-mounts were post fixed for 30 minutes in 4% paraformaldehyde/0.1% PBS and washed in 0.5% Triton X-100 in PBS. Retinas were frozen at −80° C. for 10 minutes to permeate the nuclear membrane and improve antibody permeation before blocking in 10% normal donkey serum (NDS), 2% bovine serum albumin (BSA) and 2% Triton X-100 in PBS for 60 minutes at room temperature. RGCs were counterstained with antibodies against Brn3A (1:200 Santa Cruz, #sc-31984) and visualised under fluorescence microscopy using a 20× objective and a Leica DM6000 epifluorescence microscope (Leica Microsystems, Wetzlar, Germany). Higher resolution images were be obtained using a Leica SP5 confocal microscope (Leica Microsystems) equipped with a 40× oil objective using a 1.5× digital zoom and 0.5-0.8 sequential scanning z-step interval. RGC cell counts were measured by ImageJ using the image-based tool for counting nuclei plugin (ITCN) and expressed as density of RGCs/mm².

Constructs and Vectors

The inventors have generated a genetic construct, as shown in FIG. 1, which may be used to treat a subject afflicted with an optic nerve pathology, such as glaucoma, or a cochlear pathology, or for promoting nerve regeneration and/or survival. The construct has been designed to maintain or increase the density of TrkB receptors on the cell surface of RGCs and maintain or increase signaling through the TrkB receptor pathway by concomitant production and local release of mBDNF.

The construct comprises transgenes encoding the TrkB receptor and its agonist, mature brain-derived neurotrophic factor. These transgenes are operably-linked to a single promoter, which is either the human synapsin I (SYN I) promoter or the CAG promoter. Advantageously, the construct of FIG. 1 can be placed in a rAAV2 vector without being hindered by the size of the transgenes that it encodes. This is because the construct is orientated such that the first transgene, TrkB, is linked to the viral 2A peptide sequence followed by the BDNF signal peptide and then the mature protein. This orientation also minimises immunogenicity risks because the short N-terminal amino acid sequence of the viral 2A peptide remains attached to the intracellular portion of the TrkB receptor and the residual proline amino acid from the C-terminal viral 2A sequence remains attached to the N-terminal BDNF signal peptide and is ultimately removed from the mBDNF protein following cleavage. The vector may be placed in a pharmacologically acceptable buffered solution, which may be administered to a subject.

FIGS. 2-5 show various embodiments of expression vectors. FIG. 2 shows the vector known as “Plasmid QTA001PA” containing canonical signal sequence (blue) (i.e. MTILFLTMVISYFGCMKA [SEQ ID NO:20]) plus proBDNF (red) and mBDNF (black). FIG. 3 shows the vector known as “Plasmid QTA002P”. It does not encode proBDNF but produces only mBDNF, and encodes the same signal sequence (blue) as QTA001PA. FIG. 4 shows the vector known as “Plasmid QTA003P” which also does not encode proBDNF but produces only mBDNF. Instead of the canonical signal sequence for mBDNF, it comprises an IL-2 signal sequence (blue). Finally, FIG. 5 shows the vector known as “Plasmid QTA004P”. It does not encode proBDNF but instead produces only mBDNF. It also encodes a novel signal sequence (blue), [SEQ ID NO: 32].

The inventors have produced and investigated the construct and vector relating to the glaucoma gene therapy concept starting with the mature BDNF (mBDNF) element. They have clearly demonstrated production and release of mBDNF from HEK293 cells following lipofectamine transduction with a plasmid which contains the BDNF sequence without the proBDNF coding region (QTA002P, see FIG. 3) (see FIG. 7). The mBDNF released from the cells is the predicted 14 kDa monomer (measured using Western blotting and a commercially available antibody for BDNF) and there is no evidence for protein aggregates, as has been reported by several groups attempting to generate commercial amounts of mBDNF using yeast and other cell-based manufacturing approaches¹. The mBDNF is therefore released in a form which can allow the protein molecules to form non-covalent dimers in order to activate TrkB receptors.

Using an ELISA for BDNF (which does not differentiate between mBDNF and the larger extended proBDNF protein), the inventors have also demonstrated that it is possible to substitute the DNA sequence coding for the endogenous canonical 18-amino acid signal peptide sequence (MTILFLTMVISYFGCMKA) with a novel peptide sequence (QTA004P—see FIG. 5) and release equivalent levels of BDNF into the HEK293 incubation medium following lipofectamine transduction of the cells with plasmids containing the BDNF gene (see FIG. 7).

Substitution of the endogenous signal peptide with the sequence coding for the interleukin-2 signal peptide (QTA003P—see FIG. 4) was less effective in releasing BDNF from the medium. Levels of BDNF released into the medium are currently around 1-2 nM and concentrations of this agonist are sufficient to maximally activate the specific TrkB receptors (IC50 of around 0.9 nM). Levels of BDNF release are approximately 35-fold higher (876±87 ng/mL BDNF) with the plasmid QTA001PA (see FIG. 2) which contains the combined proBDNF and mBDNF sequences and which also includes the 18-amino acid canonical signal peptide as compared to the plasmids QTA002P (see FIG. 3) and QTA004P (see FIG. 5).

Measurements of BDNF remaining in the cell by quantitative Western blotting 24 hours after lipofectamine plasmid transduction revealed lower BDNF remaining concentrations with QTA001PA than those with QTA002P and QTA004P (see FIG. 8).

Moreover, around half of the BDNF immunoreactivity in the cell lysates transduced by QTA001PA was in the form of the proBDNF (molecular weight band at 32 kDa) whereas the proBDNF band was absent in the lysates of cells transduced with QTA002P, QTA003P and QTA004P (see FIG. 9), probably because these plasmids do not contain a proBDNF extended coding sequence.

Using an ELISA specific for the proBDNF, the inventors were able to demonstrate that around 70 ng/mL (2.2 nM or 3.5%) of released BDNF-immunoreactivity from cells transduced by QTA001PA is in the form of proBDNF whilst the majority (96.5% or 876 ng/mL/63 nM) is released as mBDNF (see FIG. 10). There was no proBDNF-immunoreactivity detected from cells transduced by QTA002P, QTA003P or QTA004P which do not contain the coding sequence for the extended proBDNF.

Accordingly, it is clear that all of the plasmids are capable of producing the 14 kDa mBDNF protein, but that the amounts of mBDNF released from the HEK293 cells are largely dependent on efficiency in protein storage and packaging into secretory vesicles. The extended form of the protein, containing the combined proBDNF and mBDNF sequences, as produced with plasmid QTA001PA (FIG. 2) is therefore packaged into secretory vesicles and released into the incubation medium much more efficiently than with the smaller mBDNF sequences which appear to accumulate within the cell.

Referring to FIG. 11, it shows that substitution of the coding for the endogenous canonical signal peptide sequence, as represented in plasmid QTA002P, with novel sequences included in plasmids QTA009P to QTA013P increases the concentration of BDNF in HEK293 cells 24 hours after transduction with plasmids. FIG. 12 demonstrates that substitution of the endogenous canonical signal peptide coding sequence included in plasmid QTA002P with novel sequences (plasmids QTA009P to QTA013P) increases release of BDNF (as measured by ELISA) from HEK293 cells, as measured 24 hours after transduction with plasmids.

As shown in FIG. 13, the addition of the viral-2A peptide sequence results in efficient processing of the coding sequence for the large precursor protein into two transgenes, eGFP and BDNF. The Western blots show HEK293 cells 24 hours after they were transduced with plasmids: (i) QTA015P (expressing BDNF and eGFP separated by an IRES spacer), (ii) QTA021P (expressing BDNF followed by eGFP separated by a functional viral-2A peptide sequence), (iii) QTA022P (expressing BDNF followed by eGFP separated by a non-functional viral-2A peptide sequence) and (iv) QTA023P (expressing eGFP followed by coding for BDNF separated by a functional viral-2A peptide sequence).

The coding sequence of QTA021P (plasmid containing codon optimised sequence for mBDNF-viral-2A peptide-eGFP) is referred to here as SEQ ID No: 104, as follows:

[SEQ ID No: 104] ATGACTATCCTGTTTCTGACAATGGTTATTAGCTATTTCGGTTGCATGAA GGCTCACAGTGATCCCGCACGCCGCGGAGAACTTAGCGTGTGCGACAGCA TCAGCGAGTGGGTCACCGCCGCCGATAAGAAGACCGCTGTGGATATGTCC GGCGGGACCGTCACTGTACTCGAAAAAGTTCCAGTGAGCAAAGGCCAACT GAAACAATATTTCTATGAAACTAAGTGCAACCCCATGGGGTACACCAAGG AGGGCTGCCGGGGAATCGACAAGAGACACTGGAATTCCCAGTGCCGGACC ACTCAGAGCTACGTCCGCGCCTTGACGATGGATTCAAAGAAGCGCATCGG ATGGCGGTTCATAAGAATCGACACCAGTTGTGTGTGCACGCTGACGATAA AACGGGGGCGGGCCCCCGTGAAGCAGACCCTGAACTTTGATTTGCTCAAG TTGGCGGGGGATGTGGAAAGCAATCCCGGGCCAATGGTGAGCAAGGGCGA GGAGCTGTTCACCGGCGTTGTGCCAATACTGGTTGAGTTGGATGGCGATG TCAACGGACACAAATTTAGCGTAAGCGGGGAGGGAGAGGGCGACGCCACA TATGGCAAGCTGACCCTGAAGTTCATTTGCACGACCGGCAAATTGCCCGT CCCTTGGCCCACACTTGTGACGACCCTGACTTATGGCGTACAGTGCTTCA GCAGGTACCCTGATCATATGAAGCAACACGACTTCTTTAAGAGTGCCATG CCAGAGGGATACGTCCAGGAAAGAACCATATTCTTCAAAGATGATGGAAA TTACAAAACCCGGGCAGAGGTCAAGTTTGAAGGCGACACCCTGGTGAACA GGATCGAACTCAAAGGCATCGATTTCAAAGAGGACGGAAACATCCTCGGA CACAAACTGGAATACAATTACAACAGCCACAACGTCTACATCATGGCAGA TAAACAAAAGAACGGTATTAAAGTGAACTTCAAGATCCGGCACAACATCG AAGACGGCTCCGTCCAGCTTGCCGACCACTACCAGCAAAATACCCCGATC GGCGACGGCCCCGTTCTCCTCCCCGATAATCACTACCTGAGTACACAGTC AGCCTTGAGCAAAGACCCTAATGAAAAGCGGGACCACATGGTTTTGCTGG AGTTCGTTACCGCAGCGGGTATTACGCTGGGTATGGACGAGCTTTACAAG TAA

The coding sequence of QTA022P (plasmid containing codon optimised sequence for mBDNF-non-functional viral-2A peptide-eGFP) is referred to here as SEQ ID No: 105, as follows:

[SEQ ID No: 105] ATGACTATCCTGTTTCTGACAATGGTTATTAGCTATTTCGGTTGCATGAA GGCTCACAGTGATCCCGCACGCCGCGGAGAACTTAGCGTGTGCGACAGCA TCAGCGAGTGGGTCACCGCCGCCGATAAGAAGACCGCTGTGGATATGTCC GGCGGGACCGTCACTGTACTCGAAAAAGTTCCAGTGAGCAAAGGCCAACT GAAACAATATTTCTATGAAACTAAGTGCAACCCCATGGGGTACACCAAGG AGGGCTGCCGGGGAATCGACAAGAGACACTGGAATTCCCAGTGCCGGACC ACTCAGAGCTACGTCCGCGCCTTGACGATGGATTCAAAGAAGCGCATCGG ATGGCGGTTCATAAGAATCGACACCAGTTGTGTGTGCACGCTGACGATAA AACGGGGGCGGGCCCCTGTCAAACAAACCCTCAATTTTGACTTGCTGAAG CTTGCTGGGGATGTCGAGTCCGCTGCCGCGGCTATGGTGAGCAAGGGCGA GGAGCTGTTCACCGGCGTTGTGCCAATACTGGTTGAGTTGGATGGCGATG TCAACGGACACAAATTTAGCGTAAGCGGGGAGGGAGAGGGCGACGCCACA TATGGCAAGCTGACCCTGAAGTTCATTTGCACGACCGGCAAATTGCCCGT CCCTTGGCCCACACTTGTGACGACCCTGACTTATGGCGTACAGTGCTTCA GCAGGTACCCTGATCATATGAAGCAACACGACTTCTTTAAGAGTGCCATG CCAGAGGGATACGTCCAGGAAAGAACCATATTCTTCAAAGATGATGGAAA TTACAAAACCCGGGCAGAGGTCAAGTTTGAAGGCGACACCCTGGTGAACA GGATCGAACTCAAAGGCATCGATTTCAAAGAGGACGGAAACATCCTCGGA CACAAACTGGAATACAATTACAACAGCCACAACGTCTACATCATGGCAGA TAAACAAAAGAACGGTATTAAAGTGAACTTCAAGATCCGGCACAACATCG AAGACGGCTCCGTCCAGCTTGCCGACCACTACCAGCAAAATACCCCGATC GGCGACGGCCCCGTTCTCCTCCCCGATAATCACTACCTGAGTACACAGTC AGCCTTGAGCAAAGACCCTAATGAAAAGCGGGACCACATGGTTTTGCTGG AGTTCGTTACCGCAGCGGGTATTACGCTGGGTATGGACGAGCTTTACAAG TAA

The coding sequence of QTA023P (plasmid containing codon optimised sequence for eGFP-viral-2A peptide-mBDNF) is referred to here as SEQ ID No: 106, as follows:

[SEQ ID No: 106] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGG GCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTA CGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACC AGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAC TACCTGAGCACCCAGTCCGCCCTGAGCAAGGACCCCAACGAGAAGCGCGA TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA TGGACGAGCTGTACAAGGCTCCCGTTAAACAAACTCTGAACTTCGACCTG CTGAAGCTGGCTGGAGACGTGGAGTCCAACCCTGGACCTATGACCATCCT TTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCGCACTCCG ACCCTGCCCGCCGTGGGGAGCTGAGCGTGTGTGACAGTATTAGCGAGTGG GTCACAGCGGCAGATAAAAAGACTGCAGTGGACATGTCTGGCGGGACGGT CACAGTCCTAGAGAAAGTCCCGGTATCCAAAGGCCAACTGAAGCAGTATT TCTACGAGACCAAGTGTAATCCCATGGGTTACACCAAGGAAGGCTGCAGG GGCATAGACAAAAGGCACTGGAACTCGCAATGCCGAACTACCCAATCGTA TGTTCGGGCCCTTACTATGGATAGCAAAAAGAGAATTGGCTGGCGATTCA TAAGGATAGACACTTCCTGTGTATGTACACTGACCATTAAAAGGGGAAGA TAG

Referring to FIG. 14A, there is shown a Western blot of HEK293 cell homogenates 48 hours after transfection with the QTA020V vector. It shows efficient processing of the large precursor coding region which includes the TrkB receptor and BDNF separated by the viral-2A peptide sequence. The two TrkB and mBDNF-immunoreactive transgenes are within in the predicted correct molecular weight sizes. A lack of staining of large precursor protein above the TrkB receptor band should be noted, indicating almost complete or complete processing of the precursor protein in five repeats. FIGS. 14B and 14C show that the transgene proteins produced after vial-2A peptide cleavage have been transported to the correct intracellular compartments in HEK293 cells after processing (TrkB receptors to the cell surface and BDNF to storage vesicles prior to release).

FIG. 15 shows that addition of the viral-2A peptide sequence separating the two coding regions for the TrkB receptor and BDNF results in efficient processing into the two transgenes in mouse retina following intravitreal injection of the rAAV2 vector, QTA020V.

FIG. 16 shows the expression of transgenes in mouse retinal ganglion cell layer as shown by immunocytochemistry following injection of QTA020V, a rAAV2 vector containing the coding for the TrkB receptor and BDNF, separated by the viral-2A peptide sequence. Target retinal ganglion cell bodies are stained red with anti-Brn3A antibodies and cell nuclei are counter-stained blue with DAPI to distinguish the retinal layers.

Referring to FIG. 17, there is shown pre-treatment of QTA020V (containing coding for TrkB receptor and BDNF, separated by the viral-2A peptide sequence) via intravitreal injection (2 μl of 9×10¹² vector particles/ml) imparts significant neuroprotective efficacy on retinal ganglion cell survival following optic nerve crush in the mouse versus control animals treated with rAAV2-CAG-eGFP vector. The level of neuroprotection by the QTA020V vector was also greater than that provided by a vector expressing only BDNF. All three groups of animals were subjected to optic nerve crush procedure and the number of retinal ganglion cells measured 7 days after the insult. Retinal ganglion cells were reduced by 71% in controls (black bars) versus animals subject to sham crush (data not shown).

REFERENCES

-   1. Quigley H A, Number of people with glaucoma worldwide. Brit J.     Ophthalmol. 1996, vol. 80, PP: 389-393. -   2. www.preventblindness.org -   3. Goldberg I, Relationship between intraocular pressure and     preservation of visual field in glaucoma. Surv. Ophthalmol. 2003     vol. 48 Suppl. 1, PP: S3-S7. -   4. Glaucoma, Merck Manual of Diagnosis and Therapy, 1999, Merck     Research Laboratories; Whitehouse Station, N.J., PP: 733-738. -   5. Alward W L, Medical Management of Glaucoma. New Eng. J. Med.,     1998; vol. 339, PP: 1298-1307 -   6. Coleman A L, Glaucoma. Lancet, 1999; vol. 354, PP: 1803-1810. -   7. Medeiros F A, and Weinreb R N, Medical Backgrounders: glaucoma.     Drugs of Today 2002, vol. 38, PP: 563-570. -   8. Bakalash S, Kipnis J, Yoles E, and Schwartz M, Resistance of     retinal ganglion cells to an increase in intraocular pressure is     immune-dependent. Invest. Ophthalmol. Vis. Sci., 2002, vol. 43, PP:     2648-2653. -   9. Kipnis J, Yoles E, Porat Z, Cohen A, Mor F, Sela M, Cohen I R,     and Schwartz M, T cell immunity to copolymer 1 confers     neuroprotection on the damaged optic nerve: Possible therapy for     optic neuropathies. Proc. Natl. Acad. Sci. 2000, vol. 97, PP:     7446-7451. -   10. Quigley H A, Nickells R W, Kerrigan L A, Pease M E, Thibault D     J, and Zack D J, Retinal ganglion cell death in experimental     glaucoma and after axotomy occurs by apoptosis. Invest. Ophthalmol.     Vis. Sci. 1995 vol. 36, PP: 774-786. -   11. Weinreb R N, and Levin L A, Is neuroprotection a viable therapy     for glaucoma? Arch.

Ophthalmol. 1999, vol. 117, PP: 1540-1544.

-   12. Chao M V. Neurotrophins and their receptors: A convergence point     for many signalling pathways. Nature Rev. Neurosci. 2003, vol. 4,     PP: 299-309. -   13. Dawbarn D, and Allen S J, Neurotrophins and neurodegeneration.     Neuropathol. Appl. Neurobiol. 2003, vol. 29, PP: 211-230. -   14. Barde Y-A, Leibrock J, Lottspeich F, Edgar D, Yancopoulos G, and     Thoenen H, Brain-derived neurotrophic factor 1993, U.S. patent Ser.     No. 05/229,500. -   15. Mey J, and Thanos S, Intravitreal injections of neurotrophic     factors support the survival of axotomized retinal ganglion cells in     adult rats in vivo. Brain Res. 1993, 602: 304-317. -   16. Mansour-Rabaey S, Clarke D B, Wang Y-C, Bray G M, and Aguayo A     J, Effects of ocular injury and administration of brain-derived     neurotrophic factor on survival and regrowth of axotomized retinal     ganglion cells. Proc. Natl. Acad. Sci. USA. 1994, vol. 91, PP:     1632-1636. -   17. Peinado-Ramon P, Salvador M, Vi{umlaut over (υ)}egas-Perez M P,     and Vidal-Sanz M, Effects of axotomy and intraocular administration     of NT-4, NT-3 and brain-derived neurotrophic factor on the survival     of adult rat retinal ganglion cells. A quantitative in vivo study.     Invest Ophthalmol. Vis. Sci. 1996, vol. 37, PP: 489-500. -   18. Di Polo A, Aigner L J, Dunn R J, Bray G M, and Aguayo A J,     Prolonged delivery of brain-derived neurotrophic factor by     adenovirus-infected M{umlaut over (υ)}ller cells temporarily rescues     injured retinal ganglion cells. Proc. Natl. Acad. Sci. USA. 1998,     vol. 95, PP: 3978-3983. -   19. Klocker N, Kermer P, Weishaupt J H, Labes M, Ankerhold R, and     Bahr M, Brain-derived neurotrophic factor-mediated neuroprotection     of adult rat retinal ganglion cells in vivo does not exclusively     depend on phosphatidyl-inositol-3′-kinase/protein kinase B     signaling. J. Neurosci. 2000, vol. 20, PP: 6962-6967. -   20. Ko M L, Hu D N, Ritch R, Sharma S C, and Chen C F, Patterns of     retinal ganglion cell survival after brain-derived neurotrophic     factor administration in hypertensive eyes of rats. Neurosci. Lett.     2001, vol. 305, PP: 139-142. -   21. Chen H, and Weber A J, BDNF enhances retinal ganglion cell     survival in cats with optic nerve damage. Invest Opthamol. Vis. Sci.     2001, vol. 42, PP: 966-974. -   22. P     rez M T R, and Caminos E, Expression of brain-derived neurotrophic     factor and its functional receptor in neonatal and adult rat retina.     Neurosci. Lett. 1995, vol. 183, PP: 96-99. -   23. Vecino E, Ugarte M, Nash M S, and Osborne N N. NMDA induces BDNF     expression in the albino rat retina in vivo. Neuroreport. 1999 vol.     10, PP: 1103-1106. -   24. Mowla S J, Farhadi H F, Pareek S, Atwal J K, Morris S J, Seidah     N G, and Murphy R A.

Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J. Biol. Chem. 2001 vol 276, PP: 12660-12666.

-   25. Gupta V K, You Y, Gupta V B, Klistorner A, and Graham S L. TrkB     receptor signalling: Implications in neurodegenerative, psychiatric     and proliferative disorders. Int. J. Mol. Sci. 2013, vol. 14, PP:     10122-10142 -   26. Teng, H. K., Teng, K. K., Lee, R., Wright, S., Tevar, S.,     Almeida, R. D., Kermani, P., Torkin, R., Chen, Z. Y., Lee, F. S.,     Kraemer, R. T., Nykjaer, A. and Hempstead, B. L. ProBDNF induces     neuronal apoptosis via activation of a receptor complex of p75NTR     and sortilin. J. Neurosci. 2005, vol. 25, PP: 5455-5463. -   27. Wei Y, Zhang F, Zao J, Jiang X, Lu Q, Gao E and Wand N. Enhanced     protein expression of proBDNF and proNGF in elevated intraocular     pressure-induced rat retinal ischemia. Chin. Med. J. 2012, vol. 125,     PP: 3875-3879. -   28. Woo N H, Teng H K, Siao C-J, Chiaruttini C, Pang P T, Milner T     A, Hempstead B L and Lu B. Activation of p75^(NTR) by proBDNF     facilitates hippocampal long-term depression. Nature Neurosci. 2005,     vol 8, PP: 1069-1077. -   29. Lebrun-Julien F, Bertrand M J, De Backer O, Stellwagen D,     Morales C R, Di Polo A, and Barker P A. ProNGF induces     TNFalpha-dependent death of retinal ganglion cells through a p75NTR     non-cell-autonomous signaling pathway. Proc. Natl. Acad. Sci. USA.     2010 vol. 107, PP: 3817-3822. -   30. Quigley H A, McKinnon S J, Zack D J, Pease M E,     Kerrigan-Baumrind L A, Kerrigan D F, and Mitchell R S, Retrograde     axonal transport of BDNF in retinal ganglion cells is blocked by     acute IOP elevation in rats. Invest. Ophthalmol. Vis. Sci., 2000     vol. 41, PP: 3460-3466. -   31. Pease M E McKinnon S J, Quigley H A, Kerrigan-Baumrind L A, and     Zack D J, Obstructed axonal transport of BDNF and its receptor TRKB     in experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 2000, vol.     41, PP: 764-774. -   32. Wei Y, Wang N, Lu Q, Zhang N, Zheng D, and Li J. Enhanced     protein expressions of sortilin and p75NTR in retina of rat     following elevated intraocular pressure-induced retinal ischemia.     Neurosci. Lett. 2007, vol. 429, PP: 169-174. -   33. Martin K R G, Quigley H A, Zack D J, Levkovitch-Verbin H,     Kielczewski J, Valenta D, Baumrind L, Pease M E, Klein R L, and     Hauswirth W W, Gene therapy with brain-derived neurotrophic factor     as a protection: Retinal ganglion cells in a rat glaucoma model.     Invest. Ophthalmol. Vis. Sci., 2003, vol. 44, PP: 4357-4365. -   34. Ren R, Li Y, Liu Z, Liu K, and He S, Long-term rescue of rat     retinal ganglion cells and visual function by AAV-mediated BDNF     expression after acute elevation of intraocular pressure. Invest.     Ophthamol. Vis. Sci., 2012, vol. 53, PP: 1003-1011. -   35. Cheng L, Sapieha P, Kittlerova P, Hauswirth W W, Di Polo A, TrkB     gene transfer protects retinal ganglion cells from axotomy-induced     death in vivo. J. Neurosci., 2002, vol. 22, PP: 3977-3986. -   36. Bai Y, Xu J, Brahimi F, Zhuo Y, Sarunic M V, and Saragovi H U,     An agonistic TrkB mAb causes sustained TrkB activation, delays RGC     death, and protects the retinal structure in optic nerve axotomy and     in glaucoma. Invest. Ophthalmol. Vis. Sci. 2012, vol. 51, PP:     4722-4731. -   37. Jelsma T N, Hyman Friedman H, Berkelaar M, Bra. G M, and Aguayo     A J, Different forms of the neurotrophin receptor trkB mRNA     predominate in rat retina and optic nerve. J. Neurobiol. 1993, vol.     24, PP: 1207-1214. -   38. Gomes J R, Costa J T, Melo C V, Felizzi F, Monteiro P, Pinto M     J, Inácio A R, Wieloch T, Almeida R D, Grãos M, and Duarte C B,     Excitotoxicity down regulates TrkB.Fl signaling and up regulates the     neuroprotective truncated TrkB receptors in cultured hippocampal and     striatal neurons J. Neurosci. 2012, vol. 32, PP: 4610-4622. -   39. Gupta V K, You Y, Klistorner A, and Graham S L. Shp-2 regulates     the TrkB receptor activity in the retinal ganglion cells under     glaucomatous stress. Biochimica et Biophysica Acta 2012, vol. 1822,     PP: 1643-1649. -   40. Khalin I, Alyautdin R, Kocherga G, Bakar M A, Targeted delivery     of brain-derived neurotrophic factor for the treatment of blindness     and deafness. Int. J. Nanomedicine. 2015, vol. 10, PP: 3245-3267. -   41. Budenz C L, Wong H T, Swiderski D L, Shibata S B, Pfingst B E,     Raphael Y, Differential effects of AAV.BDNF and AAV.Ntf3 in the     deafened adult guinea pig ear. Sci. Rep. 2015, vol. 5 PP: 8619. -   42. Havenith S, Versnel H, Klis S F, Grolman W, Local delivery of     brain-derived neurotrophic factor on the perforated round window     membrane in Guinea pigs: a possible clinical application. Otol     Neurotol. 2015, vol. 36, PP: 705-711 -   43. Jian-Yi Zhang J-Y, Luo X-G, Xian C J, Liu Z-H, Zhou X-F (2008)     Endogenous BDNF is required for myelination and regeneration of     injured sciatic nerve in rodents. Eur. J. Neurosci. Vol. 12, PP:     4171-4180. -   44. Lindsey R M (1988) Nerve growth factors (NGF, BDNF) enhance     axonal regeneration but are not required for survival of adult     sensory neurons. J. Neurosci. vol. 8, PP: 2394-2405. -   45. Martinez-Salas E. Internal ribosome entry site biology and its     use in expression vectors. Curr. Opin. Biotechnol. 1999, vol. 10,     PP: 458-464. -   46. Harries M et al. Comparison of bicistronic retroviral vectors     containing internal ribosome entry sites (IRES) using expression of     human interleukin-12 (IL-12) as a readout. J. Gene Med. 200 vol. 2,     PP: 243-249. -   47. Furler S, Paterna J-C, Weibel M and Bueler H Recombinant AAV     vectors containing the foot and mouth disease virus 2A sequence     confer efficient bicistronic gene expression in cultured cells and     rat substantia nigra neurons Gene Ther. 2001, vol. 8, PP: 864-873. 

The invention claimed is:
 1. A genetic construct comprising a promoter operably linked to a first coding sequence, which encodes the tyrosine kinase receptor B (TrkB), and a second coding sequence, which encodes an agonist of the TrkB receptor, wherein the agonist is mature BDNF and wherein the genetic construct comprises a spacer sequence disposed between the first and second coding sequences, which spacer sequence encodes a peptide spacer that is configured to be digested to thereby produce the TrkB receptor and agonist as separate molecules.
 2. A genetic construct according to claim 1, wherein the promoter is the human synapsin I (SYN I) promoter or the CAG promoter.
 3. A genetic construct according to claim 1, wherein the spacer sequence comprises and encodes a viral peptide spacer sequence.
 4. A genetic construct according to claim 1, wherein the peptide spacer sequence comprises an amino acid sequence substantially as set out in SEQ ID NO. 4, or a fragment or variant thereof; or (ii) an amino acid sequence substantially as set out in SEQ ID NO. 6, or a fragment or variant thereof; or (iii) an amino acid sequence substantially as set out in SEQ ID NO. 8, or a fragment or variant thereof.
 5. A genetic construct according to claim 1, wherein the spacer sequence comprises: (i) a nucleotide sequence substantially as set out in SEQ ID NO. 5, or a fragment or variant thereof; or (ii) a nucleotide sequence substantially as set out in SEQ ID NO. 7, or a fragment or variant thereof.
 6. A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding the human canonical isoform of TrkB comprising an amino acid sequence substantially as set out in SEQ ID NO. 9, or a fragment or variant thereof, and/or wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 10, or a fragment or variant thereof.
 7. A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence which encodes isoform 4 of TrkB, and (i) wherein isoform 4 of TrkB comprises an amino acid sequence substantially as set out in SEQ ID NO. 11, or a fragment or variant thereof, and/or (ii) wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 12, or a fragment or variant thereof.
 8. A genetic construct according to claim 1, wherein the first coding sequence comprises a nucleotide sequence encoding a mutant form of TrkB receptor, wherein one or more tyrosine residue at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 is modified or mutated, and/or wherein at least two, three or four tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified, and/or wherein all five tyrosine residues at position 516, 701, 705, 706 and/or 816 of SEQ ID No: 9 are modified, and/or wherein the or each tyrosine residue is modified to a glutamic acid.
 9. A genetic construct according to claim 8, wherein the modified form of the TrkB receptor comprises an amino acid sequence substantially as set out in SEQ ID NO. 13, or a fragment or variant thereof, and/or wherein the first coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 14, or a fragment or variant thereof.
 10. A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence which encodes mature BDNF, and/or wherein mature BDNF comprises an amino acid sequence substantially as set out in SEQ ID NO. 18, or a fragment or variant thereof, and/or wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 19, or a fragment or variant thereof.
 11. A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence encoding a signal peptide for the agonist of the TrkB receptor.
 12. A genetic construct according to claim 11, wherein the second coding sequence comprises a nucleotide sequence which encodes a signal peptide comprising an amino acid sequence substantially as set out in SEQ ID NO. 20, or a fragment or variant thereof, and/or wherein the second coding sequence comprises a nucleotide sequence substantially as set out in SEQ ID NO. 21, or a fragment or variant thereof.
 13. A genetic construct according to claim 1, wherein the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide substantially as set out in any one of SEQ ID NO. 23, 25, 27 or 29, or wherein the signal peptide comprises an amino acid sequence substantially as set out in any one of SEQ ID NO. 22, 24, 26 or 28, and/or wherein the second coding sequence comprises a nucleotide sequence encoding a signal sequence peptide substantially as set out in any one of SEQ ID NO. 31, 33, 35, 37, 39, 41, 43, 45, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 or 103; or wherein the signal peptide comprises an amino acid sequence substantially as set out in any one of SEQ ID NO. 30, 32, 34, 36, 38, 40, 42, 44, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or
 102. 14. A genetic construct according to claim 1, wherein the construct comprises a nucleotide sequence substantially as set out in SEQ ID No: 107 or 108, or a fragment or variant thereof.
 15. A recombinant vector comprising the genetic construct according to claim 1, wherein the vector is a recombinant AAV (rAAV) vector, optionally wherein the rAAV is AAV-1, AAV-2, AAV-3A, AAV-3B , AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 or AAV-11.
 16. A pharmaceutical composition comprising the genetic construct according to claim 1, and a pharmaceutically acceptable vehicle.
 17. A method of treating, preventing or ameliorating an optic nerve disorder or a cochlear disorder in a subject, or for promoting nerve regeneration and/or survival in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the genetic construct according to claim
 1. 18. The method according to claim 17, wherein the optic nerve disorder that is treated is any pathophysiological condition which results in loss of RGCs, optionally wherein the optic nerve disorder that is treated is glaucoma, or wherein the cochlear disorder which is treated is hearing loss or deafness.
 19. A genetic construct according to claim 3, wherein the viral peptide spacer sequence is a viral 2A peptide spacer sequence.
 20. A genetic construct according to claim 11, wherein signal peptide for BDNF, optionally wherein the nucleotide sequence encodes the canonical signal peptide for BDNF.
 21. The method according to claim 18, wherein the pathophysiological condition is trauma to the head or face or vascular insult. 